Nucleic acid molecules encoding beta-like glycoprotein hormone polypeptide and heterodimer thereof

ABSTRACT

Novel β10 polypeptides and heterodimers thereof, and nucleic acid molecules encoding the same are disclosed. The invention also provides vectors, host cells, selective binding agents, and methods for producing β10 polypeptides and heterodimeric forms thereof, specifically α2/β10. Also provided for are methods for the treatment, diagnosis, amelioration, or prevention of diseases with β10 polypeptides and α2/β10 heterodimers or their respective binding agents.

This application is a continuation of U.S. application Ser. No.09/818,954, filed Mar. 27, 2001 now U.S. Pat. No. 7,514,239 which is acontinuation-in-part of U.S. application Ser. No. 09/723,970, filed Nov.27, 2000 now abandoned, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/199,211, filed Apr. 24, 2000, and U.S.Provisional Application Ser. No. 60/192,654, filed Mar. 28, 2000, whichare hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel beta-like member (referred toherein as “beta-10” or “β10”) of the glycoprotein hormone family andnucleic acid molecules encoding same. The invention also relates to anovel heterodimeric glycoprotein hormone comprising beta-10 and alpha-2as the subunits. The invention also relates to vectors, host cells,selective binding agents, such as antibodies, and methods for producingbeta-10 polypeptides and the disclosed beta-10 heterodimer. Alsoprovided for are methods for the use of beta-10 and the beta-10heterodimer and selective beta-10 and beta-10 heterodimer bindingagents, including methods for the diagnosis and treatment of disordersassociated with beta-10 or the beta-10 heterodimer.

BACKGROUND OF THE INVENTION

As generally accepted in the art, there are currently five knownglycoprotein hormone polypeptides produced in humans: alpha-subunit,TSH-(thyroid stimulating hormone)-β-subunit, FSH-(follicle stimulatinghormone)-β-subunit, LH-(luteinizing hormone)-β-subunit, andCG-(chorionic gonadotropin)-β-subunit; Thotakura and Blithe,Glycobiology, Volume 5, pages 3-10 (1995); Wondisford et al. in Volume1, Endocrinology (edited by L. DeGroot), pages 208-217, W. B. SaundersCompany, Philadelphia, Pa. (1995); Moyle and Campbell, in Volume 1,Endocrinology (edited by L. DeGroot), pages 230-241, W. B. SaundersCompany, Philadelphia, Pa. (1995). These polypeptides are produced bysingle genes, with the exception of the CG-β-subunit which is encoded bya multigene cluster composed of six homologous sequences linked to thesingle LH-β-subunit gene on chromosome 19; Bo and Boime, Journal ofBiological Chemistry, vol. 267, pp. 3179-3184 (1992).

Monomeric alpha-subunit (FAS, or free alpha-subunit) has hormonalactivity and is secreted by the pituitary gland and the placenta. FAShas been found to play a role in the differentiation of prolactinproducing cells in the pituitary and placenta; see Begeot et al.,Science, vol. 226, pp. 566-568 (1984), Van-Bael and Denef, Journal ofNeuroendocrinology, vol. 8, pp. 99-102 (1996), and Moy et al.,Endocrinology, vol. 137, pp. 1332-1339 (1996); and also to stimulateplacental prolactin secretion; see Blithe et al., Endocrinology, vol.129, pp. 2257-2259 (1991).

Alpha-subunit also heterodimerizes with each of the four beta-subunitsto form four heterodimeric hormones (TSH, FSH, LH and CG). TSH, FSH andLH are produced in the pituitary, stored in secretion granules, andsecreted when the appropriate releasing hormone is produced by thehypothalamus. CG is produced in the placenta and appears to be secretedconstitutively (it is not stored in secretion granules); see Wondisfordet al. in Volume 1, Endocrinology (ed. L. DeGroot), pp. 208-217, above,and Hall and Crowley, Jr. in Volume 1, Endocrinology (ed. L. DeGroot),pp. 242-258, W. B. Saunders Company, Philadelphia, Pa. (1995).

TSH influences basal metabolism by regulating the production of thyroidhormones and is used clinically for enhancing the detection andtreatment of thyroid carcinoma; see McEvoy, G. (ed.), AHFS DrugInformation, pp. 2041-2042, American Society of Health-SystemPharmacists, Inc., Bethesda, Md. (1998). In addition, diagnostic testsfor measuring TSH levels in the blood are commonly used for determiningthe functional status of the thyroid gland when thyroid gland disorderis suspected.

FSH and LH play important roles in the maintenance of reproductivefunction in males and females (i.e., gonadal maturation and gonadalsteroid production). CG is involved in the maintenance of pregnancy bystimulating the corpus luteum to produce steroid hormones during thefirst trimester. FSH, LH and CG are used clinically to treat infertilityand also as reagents in assisted reproduction procedures such as invitro fertilization (IVF); see McEvoy, G. (ed.), AHFS Drug Information,pp. 2564-2567, American Society of Health-System Pharmacists, Inc.,Bethesda, Md. (1998). Diagnostic tests for measuring FSH, LH and CGlevels are used for the diagnosis of fertility disorders, as well as totest for pregnancy.

Naturally occurring metabolites of the above mentioned glycoproteinhormone polypeptides have been described, such as the β-core fragmentwhich is derived from the beta subunit of CG, but no function has yetbeen assigned to these metabolites; Moyle and Campbell in Volume 1Endocrinology (ed. L. DeGroot) pp. 230-241, above.

In 1994, the five known glycoprotein hormone polypeptides were placedinto the cystine-knot growth factor structural superfamily, based on thecrystal structure of human CG; Lapthorn et al., Nature, vol. 369, pp.455-61 (1994). This superfamily includes the TGF-β (transforming growthfactor beta), NGF (nerve growth factor) and PDGF (platelet-derivedgrowth factor) gene families. The cystine-knot is formed by threeintramolecular disulfide bonds, has a very characteristic structure, andis responsible for the overall three-dimensional structure of all of themembers of the superfamily; Isaacs, Current Opinion in StructuralBiology, vol. 5, pp. 391-395 (1995). A recently published patentapplication describes a novel member of the cystine-knot family(zsig51); Sheppard and Lok, (1999) WIPO patent application WO99/41377.zsig51 has in fact been determined to be a new, alpha-like, member ofthe glycoprotein hormone family and will thus be referred to here as“α2” or “alpha-2” [Paszty et al. (2000) WIPO patent application WO00/78964].

SUMMARY OF THE INVENTION

The present invention provides, in part, an isolated secretable humanpolypeptide (SEQ ID NO: 1) which is a novel beta-like member of theglycoprotein hormone family and is herein designated as “beta-10” or“β10”.

The full length amino acid sequence of human β10 in accordance with thisinvention is shown in FIG. 1. The N-terminal signal peptide predictedfor the β10 polypeptide is shown underlined. The asparagine (N) atposition 87 of SEQ ID NO: 1 is located within a classic NxTglycosylation motif (where x denotes any amino acid except for prolineand T denotes threonine) and is likely to be glycosylated. The signalpeptide cleavage site in the β10 amino acid sequence is expected to bewithin the region of eight amino acids shown boxed in FIG. 1. Signalpeptide cleavage at the site which is most likely to be the authentic invivo cleavage site is reflected in the sequence of the “mature” β10polypeptide (SEQ ID NO: 3).

The most likely “mature” form (i.e., processed in situ to remove thesignal peptide) of β10 polypeptide was run against the NonRedundantProtein database using the computer analysis program known as BLAST toexamine homologies (specifically, commonly occurring or “conserved”amino acid residues) to known proteins. The top 112 “hits” were found tobe various glycoprotein hormone β-subunits from various mammalian, birdand fish species. These homologies clearly indicated that β10 is a newβ-like member of the glycoprotein hormone family.

Further, GAP analysis indicated that the homology of β10 to the fourknown human glycoprotein hormone β-subunits (mentioned above) was 31-37%identity and 42-48% similarity (see FIG. 2A-D, referred to hereinbelow).The mature forms of the four known human β glycoprotein hormonepolypeptides contain twelve cysteine residues, which form sixintramolecular disulfide bonds. The mature form of the human β10polypeptide of the present invention contains ten cysteine residues,which are likely to form five intramolecular disulfide bonds. Using thedisulfide bond cysteine pairing of CG-β as a model, the most likelydisulfide bond cysteine pairing for the five putative disulfide bonds inthe β10 polypeptide of this invention is as follows: C12-C60, C26-C75,C36-C91, C40-C93 and C96-C103 of SEQ ID NO: 3 (see also FIG. 3).

The full length amino acid sequence of mouse β10 polypeptide is setforth in SEQ ID NO: 11 and the nucleotide sequence of the full codingregion of the mouse β10 cDNA is set forth in SEQ ID NO: 12. Signalpeptide cleavage at the site which is most likely to be the authentic invivo cleavage site is reflected in the sequence of the “mature” formmouse β10 polypeptide (SEQ ID NO: 13). BestFit analysis indicated thatthe amino acid homology of mature form human β10 polypeptide as comparedto mature form mouse β10 polypeptide was 93.4% identity and 97.2%similarity (see FIG. 4, referred to herein below).

Based on the logical inclusion of the β10 polypeptide of this inventionin the glycoprotein hormone family, this polypeptide could be a monomer(analogous to FAS and β-core fragment) and/or could form a heterodimerwith one or more glycoprotein hormone family polypeptides (for exampleheterodimers α/β10, β10/TSH-β, β10/LH-β). The β10 polypeptide could alsoform heterodimers with polypeptides which are distinct from the knownglycoprotein hormone polypeptides. Based on these various possibilities,the β10 polypeptide may form more than one hormone (i.e., the β10hormones).

A heterodimerization assay was used to determine that human β10 forms aheterodimer with human α2 polypeptide, described in the above mentionedWO99/41377 and WO 00/78964 patent applications, thus discovering anddefining a novel heterodimeric glycoprotein hormone, α2/β10.

The general principle of the heterodimerization assay for secretedproteins, such as the glycoprotein hormones, is co-transfection of thetwo distinct genes into mammalian cells, collection of conditionedmedia, immunoprecipitation with an antibody that specifically binds toone of the gene products and Western blotting of the immunoprecipitatewith an antibody that specifically binds to the other gene product. Withthe proper control experiments in place, the presence of a band of thecorrect size on the Western would indicate heterodimerization of the twogene products under the experimental conditions of the assay, whereasthe absence of a band of the correct size on the Western would indicatethat the two gene products did not heterodimerize under the experimentalconditions of the assay. Because the known heterodimeric glycoproteinhormones (LH, FSH, TSH and CG) can readily be produced byco-transfection of mammalian cells with the appropriate genes, this typeof mammalian cell based co-transfection heterodimerization assay isrelevant for members of the glycoprotein hormone family.

A human α2-polyHis-tag mammalian expression vector and a humanβ10-FLAG-tag mammalian expression vector were co-transfected into 293cells and serum free conditioned media was harvested after 72 hours.Immunoprecipitation was done using anti-FLAG M2-Agarose affinity beads(Cat# A1205, Sigma, St. Louis, Mo.). A Western blot of thisimmunoprecipitate was probed with affinity purified anti-α2 rabbitpolyclonal antibodies (example 4) that had been conjugated to HorseRadish Peroxidase (Linx HRP Rapid Protein Conjugation Kit, cat#K8050-01, Invitrogen Corp., Carlsbad, Calif.). A strong α2-polyHis-tagband was observed using the ECL Western Blot detection kit (cat#RPN2106, Amersham Pharmacia Biotech, Piscataway, N.J.). Control experimentsshowed that the presence of the strong α2-polyHis-tag band on theWestern blot was entirely dependent on co-transfection with the humanβ10-FLAG-tag mammalian expression vector and the use of anti-FLAGM2-Agarose affinity beads. No α2-polyHis-tag band was observed if eitherof these 2 components was left out of the experiment or if plain agarosebeads (i.e. without anti-FLAG antibodies) were used for theimmunoprecipitation step.

Similar to the known heterodimeric glycoprotein hormones (TSH, FSH, LHand CG) α2/β10 is a heterodimer of an alpha-like glycoprotein hormonepolypeptide and a beta-like glycoprotein hormone polypeptide.

These data also indicate that recombinant, secreted α2/β10 heterodimer(without polyHis and FLAG affinity tags) can be produced in mammaliancells for various therapeutic and diagnostic utilities as describedfurther below.

Heterodimeric glycoprotein hormones such as CG can also be assembled invitro upon co-incubation of, for example, isolated alpha-subunit andisolated CG-β subunit under suitable conditions [see Blithe and Iles,Endocrinology, volume 136, pages 903-910 (1995)]. α2/β10 heterodimercould similarly be assembled in vitro upon co-incubation of isolated α2polypeptide and isolated β10 polypeptide. Such assembled α2/β10heterodimer could be used for various therapeutic and diagnosticutilities as described further below.

Transgenic mice were made that over expressed mouse α2 alone, mouse β10alone or the mouse α2/β10 heterodimer (see example 6). Only thosetransgenics over expressing the α2/β10 heterodimer showed distinctphenotypic differences as compared to control mice. The α2/β10overexpressor transgenic mice exhibited a phenotype characterized bybilateral thyroid enlargement with multiple follicular papillaryadenomas and resulting hyperthyroidism, as indicated by elevated serumT4 levels. Other phenotypic changes were felt to be related to thesystemic hyperthyroid state, and included moderate hepatomegaly,hepatocellular hyperplasia, and slightly decreased serum cholesterollevels, bilateral renal hypertrophy, and a mild to moderate leukocytosiswith a predominance of lymphocytes (see example 6). Thus in a normalmouse setting α2/β10 clearly has a thyroid stimulating hormone (TSH)like activity. Due to the high level of amino acid conservation betweenmouse α2 and human α2 [88.5% identity and 90.4% similarity for thepredicted mature forms (i.e. without signal peptide)], the high level ofamino acid conservation between mouse β10 and human β10 [93.4% identityand 97.2% similarity for the predicted mature forms (i.e. without signalpeptide)], and the very high level of similarity between mouse thyroidgland biology and human thyroid gland biology, it is anticipated thathuman α2/β10 heterodimer has the same thyroid stimulating hormone (TSH)like activity as that found for the mouse α2/β10 heterodimer. Inaddition to TSH-like activity, α2/β10 may have other, distinct,biological effects in different physiological settings (i.e., diseaseconditions), as described in greater detail further herein.

TSH influences basal metabolism by regulating the production of thyroidhormones and is used clinically for enhancing the detection andtreatment of thyroid carcinoma; see McEvoy, G. (ed.), AHFS DrugInformation, pp. 2041-2042, American Society of Health-SystemPharmacists, Inc., Bethesda, Md. (1998). In addition, diagnostic testsfor measuring TSH levels in the blood are commonly used for determiningthe functional status of the thyroid gland when thyroid gland disorderis suspected. It is likely that human α2/β10 will have similar clinicalutilities as TSH and will be useful for the treatment and diagnosis ofthyroid gland related diseases and disorders. In addition, human α2/β10may have other therapeutic and diagnostic uses which are describedherein. It is reasonable to surmise that human α2/β10 selective bindingagents, for example, antibodies, will have similar clinical utilities toTSH selective binding agents and will therefore be useful for thetreatment and diagnosis of thyroid gland related diseases and disorders.In addition, human α2/β10 selective binding agents may have othertherapeutic and diagnostic uses as described herein.

This invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO: 2;

(b) a nucleotide sequence encoding the polypeptide set forth in SEQ IDNO: 1;

(c) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of (a) or (b), wherein theencoded polypeptide, when heterodimerized to human α2 polypeptide, hasan activity of the human α2/β10 heterodimer; and

(d) a nucleotide sequence complementary to any of (a)-(c).

The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide that is at least about70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to thepolypeptide as set forth in SEQ ID NO: 1, wherein the polypeptide, whenheterodimerized to human α2 polypeptide, has an activity of the humanα2/β10 heterodimer;

(b) a nucleotide sequence encoding an allelic variant or splice variantof the nucleotide sequence set forth in SEQ ID NO: 2, wherein theencoded polypeptide, when heterodimerized to human α2 polypeptide, hasan activity of the human α2/β10 heterodimer;

(c) a nucleotide sequence of SEQ ID NO: 2, (a), or (b) encoding apolypeptide fragment of at least about 25 amino acid residues, whereinthe polypeptide, when heterodimerized to human α2 polypeptide, has anactivity of the human α2/β10 heterodimer;

(d) a nucleotide sequence of SEQ ID NO: 2 or (a)-(c) comprising afragment of at least about 16 nucleotides;

(e) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(d), wherein theencoded polypeptide, when heterodimerized to human α2 polypeptide, hasan activity of the human α2/β10 heterodimer; and

(f) a nucleotide sequence complementary to any of (a)-(d).

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 1 with at least one conservative amino acid substitution, whereinthe polypeptide, when heterodimerized to human α2 polypeptide, has anactivity of the human α2/β10 heterodimer;

(b) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 1 with at least one amino acid insertion, wherein the polypeptide,when heterodimerized to human α2 polypeptide, has an activity of thehuman α2/β10 heterodimer;

(c) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 1 with at least one amino acid deletion, wherein the polypeptide,when heterodimerized to human α2 polypeptide, has an activity of thehuman α2/β10 heterodimer;

(d) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 1 which has a C- and/or N-terminal truncation, wherein thepolypeptide, when heterodimerized to human α2 polypeptide, has anactivity of the human α2/β10 heterodimer;

(e) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 1 with at least one modification selected from the group consistingof amino acid substitutions, amino acid insertions, amino aciddeletions, C-terminal truncation, and N-terminal truncation, wherein thepolypeptide, when heterodimerized to human α2 polypeptide, has anactivity of the human α2/β10 heterodimer;

(f) a nucleotide sequence of (a)-(e) comprising a fragment of at leastabout 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(f), wherein theencoded polypeptide, when heterodimerized to human α2 polypeptide, hasan activity of the human α2/β10 heterodimer; and

(h) a nucleotide sequence complementary to any of (a)-(e).

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

(a) the mature amino acid sequence set forth in SEQ ID NO: 3, andoptionally further comprising an amino-terminal methionine;

(b) an amino acid sequence for an ortholog of SEQ ID NO: 3, wherein theencoded polypeptide, when heterodimerized to human α2 polypeptide, hasan activity of the human α2/β10 heterodimer;

(c) an amino acid sequence that is at least about 70, 75, 80, 85, 90,95, 96, 97, 98, or 99 percent identical to the amino acid sequence ofSEQ ID NO: 3, wherein the polypeptide, when heterodimerized to human α2polypeptide, has an activity of the human α2/β10 heterodimer;

(d) a fragment of the amino acid sequence set forth in SEQ ID NO: 3comprising at least about 25 amino acid residues, wherein thepolypeptide, when heterodimerized to human α2 polypeptide, has anactivity of the human α2/β10 heterodimer;

(e) an amino acid sequence for an allelic variant or splice variant ofeither the amino acid sequence as set forth in SEQ ID NO: 3, or at leastone of (a)-(c) wherein the polypeptide, when heterodimerized to human α2polypeptide, has an activity of the human α2/β10 heterodimer.

The invention further provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in SEQ ID NO: 3 with at leastone conservative amino acid substitution, wherein the polypeptide, whenheterodimerized to human α2 polypeptide, has an activity of the humanα2/β10 heterodimer;

(b) the amino acid sequence as set forth in SEQ ID NO: 3 with at leastone amino acid insertion, wherein the polypeptide, when heterodimerizedto human α2 polypeptide, has an activity of the human α2/β10heterodimer;

(c) the amino acid sequence as set forth in SEQ ID NO: 3 with at leastone amino acid deletion, wherein the polypeptide, when heterodimerizedto human α2 polypeptide, has an activity of the human α2/β10heterodimer;

(d) the amino acid sequence as set forth in SEQ ID NO: 3 which has a C-and/or N-terminal truncation, wherein the polypeptide, whenheterodimerized to human α2 polypeptide, has an activity of the humanα2/β10 heterodimer; and

(e) the amino acid sequence as set forth in SEQ ID NO: 3, with at leastone modification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the polypeptide, whenheterodimerized to human α2 polypeptide, has an activity of the humanα2/β10 heterodimer.

Also provided are fusion polypeptides comprising the amino acidsequences of (a)-(e) above.

The present invention also provides for an expression vector comprisingthe isolated nucleic acid molecules as set forth herein, recombinanthost cells comprising recombinant nucleic acid molecules as set forthherein, and a method of producing a β10 polypeptide or an α2/β10heterodimer of this invention comprising culturing the host cells andoptionally isolating the β10 polypeptide or α2/β10 heterodimer soproduced.

A transgenic non-human animal comprising a nucleic acid molecule(s)encoding a β10 polypeptide or α2/β10 heterodimer of this invention isalso encompassed by the invention. The nucleic acid molecules areintroduced into the animal in a manner that allows expression andincreased levels of β10 polypeptide or α2/β10 heterodimer, which mayinclude increased circulating levels. The transgenic non-human animal ispreferably a mammal.

Also provided are derivatives of the β10 polypeptide or α2/β10heterodimer of the present invention.

Additionally provided are selective binding agents such as antibodiesand peptides capable of specifically binding the β10 polypeptide orα2/β10 heterodimer of the invention. Such antibodies and peptides may beagonistic or antagonistic to an activity of the β10 polypeptide orα2/β10 heterodimer.

Pharmaceutical compositions comprising the nucleotides, β10 polypeptideor α2/β10 heterodimer, or selective binding agents of the presentinvention and one or more pharmaceutically acceptable formulation agentsare also encompassed by the invention. The pharmaceutical compositionsare used to provide therapeutically effective amounts of the nucleotidesor polypeptides of the present invention. The invention is also directedto methods of using the nucleic acid molecules, β10 polypeptide, α2/β10heterodimer and selective binding agents.

The nucleic acid molecules, β10 polypeptide, α2/β10 heterodimer andselective binding agents of the present invention may be used to treat,prevent, ameliorate, and/or detect diseases and disorders, includingthose recited herein.

The present invention also provides a method of assaying test moleculesto identify a test molecule which binds to a β10 polypeptide or α2/β10heterodimer. The method comprises contacting the β10 polypeptide orα2/β10 heterodimer with a test molecule and determining the extent ofbinding of the test molecule to the β10 polypeptide or α2/β10heterodimer. The method further comprises determining whether such testmolecules are agonists or antagonists of the β10 polypeptide or α2/β10heterodimer. The present invention further provides a method of testingthe impact of molecules on the expression of the β10 polypeptide orα2/β10 heterodimer or on the activity of the β10 polypeptide or α2/β10heterodimer.

Methods of regulating expression and modulating (i.e., increasing ordecreasing) levels of a β10 polypeptide or α2/β10 heterodimer of thisinvention are also encompassed by the invention. One method comprisesadministering to an animal a nucleic acid molecule(s) encoding such aβ10 polypeptide or α2/β10 heterodimer. In another method, a nucleic acidmolecule comprising elements that regulate or modulate expression of theβ10 polypeptide or α2/β10 heterodimer of this invention may beadministered. Examples of these methods include gene therapy, celltherapy, and anti-sense therapy as further described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts in linear array the full coding region of human β10polypeptide in accordance with this invention (SEQ ID NO: 1). Thepredicted signal peptide region is underlined and the region containingthe predicted signal peptide cleavage site is boxed. The asparagine (N)residue that is located within the classic NxT glycosylation motif, andwhich is very likely to be glycosylated, is shown in larger font. Thecorresponding nucleic acid sequence which encodes this polypeptide (SEQID NO: 2) comprises nucleotides 1-390, inclusive, of the nucleic acidsequence shown in this Figure.

FIG. 2A-2D illustrates the relatedness of the known human glycoproteinhormone β-subunit polypeptides (prior art) and the β10 polypeptide ofthis invention. The mature form of β10 used for these comparisons (SEQID NO: 3) most likely represents the authentic in vivo form of β10polypeptide. FIGS. 2A-D comprise the GAP output showing the amino acidhomology between the mature form of β10 and respectively, TSH-(thyroidstimulating hormone)-β-subunit, FSH-(follicle stimulatinghormone)-β-subunit, LH-(luteinizing hormone)-β-subunit, andCG-(chorionic gonadotropin)-β-subunit.

FIG. 3 shows the likely disulfide bond cysteine (C) pairs of the fiveputative disulfide bonds in the most likely mature form of human β10(SEQ ID NO: 3). The ten cysteine residues are shown in large font andthe disulfide bonds are drawn as solid lines. The three disulfide bonds(C12-C60, C36-C91, C40-C93) that form the cystine-knot are drawn abovethe amino acid sequence, and the two additional disulfide bonds(C26-C75, C96-C103) are drawn below the amino acid sequence.

FIG. 4 is the BestFit output showing the amino acid homology between themature form of human β10 and the mature form of mouse β10. The matureform of human β10 used for this comparison (SEQ ID NO: 3) most likelyrepresents the authentic in vivo form of human β10 polypeptide. Themature form of mouse β10 used for this comparison (SEQ ID NO: 13) mostlikely represents the authentic in vivo form of mouse β10 polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein.

DEFINITIONS

The terms “β10 gene” or “β10 nucleic acid molecule” or “polynucleotide”refers to a nucleic acid molecule comprising or consisting of anucleotide sequence as set forth in SEQ ID NO: 2, a nucleotide sequenceencoding the polypeptide as set forth in SEQ ID NO: 1, a nucleotide ofthe DNA insert in ATCC deposit no. PTA-1210, and nucleic acid moleculesas defined herein.

The term “β10 polypeptide” refers to a polypeptide comprising the aminoacid sequence of SEQ ID NO: 3, and related polypeptides. Relatedpolypeptides include: β10 polypeptide allelic variants, β10 polypeptideorthologs, β10 polypeptide splice variants, β10 polypeptide variants andβ10 polypeptide derivatives. β10 polypeptides may be maturepolypeptides, as defined herein, and may or may not have an aminoterminal methionine residue, depending on the method by which they areprepared.

The term “β10 polypeptide allelic variant” refers to one of severalpossible naturally occurring alternate forms of a gene occupying a givenlocus on a chromosome of an organism or a population of organisms.

The term “β10 polypeptide derivatives” refers to the polypeptide setforth in SEQ ID NO: 3, polypeptide allelic variants thereof, polypeptideorthologs thereof, polypeptide splice variants thereof, or polypeptidevariants thereof, as defined herein, that have been chemically modified.

The term “β10 polypeptide fragment” refers to a polypeptide thatcomprises a truncation at the amino terminus (with or without a leadersequence) and/or a truncation at the carboxy terminus of the polypeptideset forth in SEQ ID NO: 3, polypeptide allelic variants thereof,polypeptide orthologs thereof, polypeptide splice variants thereofand/or a polypeptide variant thereof having one or more amino acidadditions or substitutions or internal deletions (wherein the resultingpolypeptide is at least 6 amino acids or more in length) as compared tothe β10 polypeptide amino acid sequence set forth in SEQ ID NO: 3.Polypeptide fragments may result from alternative RNA splicing or fromin vivo protease activity. In preferred embodiments, truncationscomprise about 10 amino acids, or about 20 amino acids, or about 50amino acids, or about 75 amino acids, or about 100 amino acids, or morethan about 100 amino acids. The polypeptide fragments so produced willcomprise about 25 contiguous amino acids, or about 50 amino acids, orabout 75 amino acids, or about 100 amino acids, or about 150 aminoacids, or about 200 amino acids. Such polypeptide fragments mayoptionally comprise an amino terminal methionine residue. It will beappreciated that such fragments can be used, for example, to generateantibodies to β10 polypeptide or α2/β10 heterodimer.

The term “β10 fusion polypeptide” refers to a fusion of one or moreamino acids (such as a heterologous peptide or polypeptide) at the aminoor carboxy terminus of the polypeptide set forth in SEQ ID NO: 3,polypeptide allelic variants, polypeptide orthologs, polypeptide splicevariants, or polypeptide variants having one or more amino aciddeletions, substitutions or internal additions as compared to thepolypeptide amino acid sequence set forth in SEQ ID NO: 3.

The term “β10 polypeptide ortholog” refers to a polypeptide from anotherspecies that corresponds to the β10 polypeptide amino acid sequence setforth in SEQ ID NO: 3. For example, mouse and human β10 polypeptides areconsidered orthologs of each other.

The term “β10 polypeptide splice variant” refers to a nucleic acidmolecule, usually RNA, which is generated by alternative processing ofintron sequences in an RNA transcript of the β10 polypeptide amino acidsequence set forth in SEQ ID NO: 3.

The term “β10 polypeptide variants” refers to β10-like polypeptidescomprising amino acid sequences having one or more amino acid sequencesubstitutions, deletions (such as internal deletions and/or polypeptidefragments), and/or additions (such as internal additions and/or fusionpolypeptides) as compared to the β10 polypeptide amino acid sequence setforth in SEQ ID NO: 3. Variants may be naturally occurring (e.g.,β10-like polypeptide allelic variants, polypeptide orthologs andpolypeptide splice variants) or artificially constructed. Suchpolypeptide variants may be prepared from the corresponding nucleic acidmolecules having a DNA sequence that varies accordingly from the DNAsequence set forth in SEQ ID NO: 2. In preferred embodiments, thevariants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75,or from 1 to 100, or more than 100 amino acid substitutions, insertions,additions and/or deletions, wherein the substitutions may beconservative, or non-conservative, or any combination thereof.

The term “α2/β10 heterodimer” refers to a heterodimer of the β10polypeptide and the α2 polypeptide.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “biologically active β10 polypeptides” refers to β10-likepolypeptides that, when heterodimerized to human α2 polypeptide, have anactivity of the human α2/β10 heterodimer.

The terms “effective amount” and “therapeutically effective amount” eachrefer to the amount of a β10 nucleic acid molecule or a β10 polypeptideor α2/β10 heterodimer of this invention used to support an observablelevel of one or more biological activities of the α2/β10 heterodimerdescribed herein.

The term “expression vector” refers to a vector which is suitable foruse in a host cell and contains nucleic acid sequences which directand/or control the expression of heterologous nucleic acid sequences.Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecules or polypeptides, as the case may be, asdetermined by the match between strings of two or more nucleotide or twoor more amino acid sequences. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity”, refers to a measure of similarity which includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, 10/20 identical amino acids,and the remainder are all non-conservative substitutions, then thepercent identity and similarity would both be 50%. If in the sameexample, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15/20). Therefore, in cases where there areconservative substitutions, the degree of similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that is free from at least one contaminatingnucleic acid molecule with which it is naturally associated. Preferably,the isolated nucleic acid molecule of the present invention issubstantially free from any other contaminating nucleic acid molecule(s)or other contaminants that are found in its natural environment whichwould interfere with its use in polypeptide production or itstherapeutic, diagnostic, prophylactic or research use.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that is free from at least one contaminating polypeptide orother contaminants that are found in its natural environment.Preferably, the isolated polypeptide is substantially free from anyother contaminating polypeptides or other contaminants that are found inits natural environment which would interfere with its therapeutic,diagnostic, prophylactic or research use.

The term “mature β10 polypeptide” refers to a β10 polypeptide lacking aleader sequence. A mature β10 polypeptide may also include othermodifications such as proteolytic processing of the amino terminus (withor without a leader sequence) and/or the carboxy terminus, cleavage of asmaller polypeptide from a larger precursor, N-linked and/or O-linkedglycosylation, and the like.

The term “nucleic acid sequence” or “nucleic acid molecule” refers to aDNA or RNA sequence. The term encompasses molecules formed from any ofthe known base analogs of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The term “operably linked” is used herein to refer to an arrangement offlanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Thus, aflanking sequence operably linked to a coding sequence may be capable ofeffecting the replication, transcription and/or translation of thecoding sequence. For example, a coding sequence is operably linked to apromoter when the promoter is capable of directing transcription of thatcoding sequence. A flanking sequence need not be contiguous with thecoding sequence, so long as it functions correctly. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more formulationmaterials suitable for accomplishing or enhancing the delivery of a β10nucleic acid molecule, β10 polypeptide, α2/β10 heterodimer or selectivebinding agents of the present invention as a pharmaceutical composition.

The term “selective binding agent” refers to a molecule or moleculeshaving specificity for the β10 polypeptide and/or α2/β10 heterodimer ofthis invention. As used herein, the terms, “specific” and “specificity”refer to the ability of the selective binding agents to bind to humanβ10 polypeptide and/or α2/β10 heterodimer and not to bind to humannon-β10 polypeptide and/or non-α2/β10 heterodimer. It will beappreciated, however, that the selective binding agents may also bindorthologs of the polypeptide set forth in SEQ ID NO: 3, and/or orthologsof human α2/β10 heterodimer, that is, interspecies versions thereof,such as mouse and rat polypeptides.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, for example, Graham et al., Virology, 52:456 (1973);Sambrook et al., Molecular Cloning, a laboratory Manual, Cold SpringHarbor Laboratories (New York, 1989); Davis et al., Basic Methods inMolecular Biology, Elsevier, 1986; and Chu et al., Gene, 13:197 (1981).Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, may be maintained transiently as an episomal element without beingreplicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

It is understood that related nucleic acid molecules include allelic orsplice variants of the nucleic acid molecule of SEQ ID NO: 2, andinclude sequences which are complementary to any of the above nucleotidesequences. Related nucleic acid molecules also include a nucleotidesequence encoding a polypeptide comprising or consisting essentially ofa substitution, modification, addition and/or a deletion of one or moreamino acid residues compared to the polypeptide in SEQ ID NO: 1.

Fragments include nucleic acid molecules which encode a polypeptide ofat least about 25 amino acid residues, or about 50, or about 75, orabout 100, or greater than about 100 amino acid residues of thepolypeptide of SEQ ID NO: 1.

In addition, related β10 nucleic acid molecules include those moleculeswhich comprise nucleotide sequences which hybridize under moderately orhighly stringent conditions as defined herein with the fullycomplementary sequence of the nucleic acid molecule of SEQ ID NO: 2, orof a molecule encoding a polypeptide, which polypeptide comprises theamino acid sequence of SEQ ID NO: 1, or of a nucleic acid fragment asdefined herein, or of a nucleic acid fragment encoding a polypeptide asdefined herein. Hybridization probes may be prepared using the β10sequences provided herein to screen cDNA, genomic or synthetic DNAlibraries for related sequences. Regions of the DNA and/or amino acidsequence of the β10 polypeptide that exhibit significant identity toknown sequences are readily determined using sequence alignmentalgorithms as described herein and those regions may be used to designprobes for screening.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015M sodium chloride, 0.0015M sodiumcitrate at 65-68° C. or 0.015M sodium chloride, 0.0015M sodium citrate,and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory,(Cold Spring Harbor, N.Y. 1989); Anderson et al., Nucleic AcidHybridisation: a practical approach, Ch. 4, IRL Press Limited (Oxford,England).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also be used,however, the rate of hybridization will be affected. Other agents may beincluded in the hybridization and washing buffers for the purpose ofreducing non-specific and/or background hybridization. Examples are 0.1%bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate (NaDodSO₄ or SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or othernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4, however, at typical ionic strength conditions, therate of hybridization is nearly independent of pH. See Anderson et al.,Nucleic Acid Hybridisation: a Practical Approach, Ch. 4, IRL PressLimited (Oxford, England).

Factors affecting the stability of a DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:T _(m)(° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−600/N−0.72(% formamide)where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately stringent conditions” refers to conditions underwhich a DNA duplex with a greater degree of base pair mismatching thancould occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015M sodiumchloride, 0.0015M sodium citrate at 50-65° C. or 0.015M sodium chloride,0.0015M sodium citrate, and 20% formamide at 37-50° C. By way ofexample, a “moderately stringent” condition of 50° C. in 0.015 M sodiumion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature raisethe ionic strength.

A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20 nucleotides is given by:T _(m)=2° C. per A-T base pair+4° C. per G-C base pair*The sodium ion concentration in 6× salt sodium citrate (SSC) is 1M. SeeSuggs et al., Developmental Biology Using Purified Genes, p. 683, Brownand Fox (eds.) (1981).

High stringency washing conditions for oligonucleotides are usually at atemperature of 0-5° C. below the Tm of the oligonucleotide in 6×SSC,0.1% SDS.

In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is about 70 percent identical tothe nucleotide sequence of SEQ ID NO: 2, or comprise or consistessentially of a nucleotide sequence encoding a polypeptide that isabout 70 percent identical to the polypeptide of SEQ ID NO: 1. Inpreferred embodiments, the nucleotide sequences are about 70 percent, 75percent, 80 percent, or about 85 percent, or about 90 percent, or about95, 96, 97, 98, or 99 percent identical to the nucleotide sequence asshown in SEQ ID NO: 2, or the nucleotide sequences encode a polypeptidethat is about 70 percent, 75 percent, 80 percent, or about 85 percent,or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical tothe polypeptide sequence as set forth in SEQ ID NO: 1.

Differences in the nucleic acid sequence may result in conservativeand/or non-conservative modifications of the amino acid sequencerelative to the amino acid sequence of SEQ ID NO: 1.

Conservative modifications to the amino acid sequence of SEQ ID NO: 1(and the corresponding modifications to the encoding nucleotides) willproduce β10-like polypeptides in accordance with this invention havingfunctional and chemical characteristics similar to those of thenaturally occurring β10 polypeptide hereof. In contrast, substantialmodifications in the functional and/or chemical characteristics of theβ10 polypeptide may be accomplished by selecting substitutions in theamino acid sequence of SEQ ID NO: 1 that differ significantly in theireffect on maintaining (a) the structure of the molecular backbone in thearea of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Conservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues may be divided into classes based on commonside chain properties:

-   1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;-   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;-   3) acidic: Asp, Glu;-   4) basic: His, Lys, Arg;-   5) residues that influence chain orientation: Gly, Pro; and-   6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the human β10polypeptide that are homologous with non-human β10 polypeptideorthologs, or into the non-homologous regions of the molecule.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.One may also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the β10polypeptide or to increase or decrease the affinity of the β10polypeptides or the α2/β10 heterodimers described herein.

Exemplary amino acid substitutions are set forth in Table I.

TABLE I Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe,Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4Diamino- Arg butyric Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala,Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptides of SEQ ID NOs: 1 or 3 using well known techniques. Foridentifying suitable areas of the molecule that may be changed withoutdestroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of a β10 polypeptide to such similar polypeptides. With such acomparison, one can identify residues and portions of the molecules thatare conserved among similar polypeptides. It will be appreciated thatchanges in areas of a β10 polypeptide that are not conserved relative tosuch similar polypeptides would be less likely to adversely affect thebiological activity and/or structure of the β10 polypeptide or theα2/β10 heterodimer. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity (conservative amino acid residue substitutions). Therefore,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a β10 polypeptide thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of the β10 polypeptide.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of that information, one skilled in the art maypredict the alignment of amino acid residues of a β10 polypeptide withrespect to its three dimensional structure. One skilled in the art maychoose not to make radical changes to amino acid residues predicted tobe on the surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. The variants can thenbe screened using activity assays know to those skilled in the art. Suchvariants could be used to gather information about suitable variants.For example, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-9 (1996)), “profile analysis” (Bowie et al., Science,253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990);Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and“evolutionary linkage” (See Home, supra, and Brenner, supra).

Preferred β10 polypeptide or α2/β10 heterodimer variants in accordancewith this invention include glycosylation variants wherein the numberand/or type of glycosylation sites has been altered compared to theamino acid sequence set forth in SEQ ID NO: 1. In one embodiment, β10polypeptide or α2/β10 heterodimer variants according to this inventioncomprise a greater or a lesser number of N-linked glycosylation sitesthan the amino acid sequence set forth in SEQ ID NO: 1. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution(s) of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionswhich eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred β10polypeptide or α2/β10 heterodimer variants include cysteine variants,wherein one or more cysteine residues are deleted from or substitutedfor another amino acid (e.g., serine) as compared to the amino acidsequence set forth in SEQ ID NO: 1. Cysteine variants are useful whenthe β10 polypeptide or α2/β10 heterodimer must be refolded into abiologically active conformation such as after the isolation ofinsoluble inclusion bodies. Cysteine variants generally have fewercysteine residues than the native protein, and typically have an evennumber to minimize interactions resulting from unpaired cysteines.

In addition, the polypeptide comprising the amino acid sequence of SEQID NO: 3 or a polypeptide variant thereof may be fused to a heterologouspolypeptide, such as but not limited to α2, to form a heterodimer.Heterologous peptides and polypeptides include, but are not limited to:an epitope to allow for the detection and/or isolation of a β10 fusionpolypeptide or an α2/β10 heterodimer; a transmembrane receptor proteinor a portion thereof, such as an extracellular domain, or atransmembrane and intracellular domain; a ligand or a portion thereofwhich binds to a transmembrane receptor protein; an enzyme or portionthereof which is catalytically active; a polypeptide or peptide whichpromotes oligomerization, such as a leucine zipper domain; a polypeptidewith which β10 normally dimerizes, such as α2; a polypeptide or peptidewhich increases stability, such as an immunoglobulin constant region;and a polypeptide which has a therapeutic activity different from thepolypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3or a polypeptide variant thereof.

Fusions can be made either at the amino terminus or at the carboxyterminus of the polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 3 or a polypeptide variant. Fusions may be direct with nolinker or adapter molecule or indirect using a linker or adaptermolecule. A linker or adapter molecule may be one or more amino acidresidues, typically up to about 20 to about 50 amino acid residues. Alinker or adapter molecule may also be designed with a cleavage site fora DNA restriction endonuclease or for a protease to allow for theseparation of the fused moieties. It will be appreciated that onceconstructed, the fusion polypeptides can be derivatized according to themethods described herein.

In a further embodiment of the invention, the polypeptide comprising theamino acid sequence of SEQ ID NO: 3 or a polypeptide variant is fused toone or more domains of an Fc region of human IgG. Antibodies comprisetwo functionally independent parts, a variable domain known as “Fab”,which binds antigen, and a constant domain known as “Fc”, which isinvolved in effector functions such as complement activation and attackby phagocytic cells. An Fc has a long serum half-life, whereas an Fab isshort-lived. Capon et al., Nature, 337:525-31 (1989). When constructedtogether with a therapeutic protein, an Fc domain can provide longerhalf-life or incorporate such functions as Fc receptor binding, proteinA binding, complement fixation and perhaps even placental transfer. Id.Table II summarizes the use of certain Fc fusions known in the art.

TABLE II Fc Fusion with Therapeutic Proteins Form of Fusion TherapeuticFc partner implications Reference IgG1 N-terminus Hodgkin's U.S. Pat.No. of CD30-L disease; 5,480,981 anaplastic lymphoma; T-cell leukemiaMurine IL-10 anti- Zheng et al. Fcγ2a inflammatory; (1995), J.transplant Immunol., 154: rejection 5590-5600 IgG1 TNF septic shockFisher et al. receptor (1996), N. Engl. J. Med., 334: 1697-1702; Van Zeeet al., (1996), J. Immunol., 156: 2221-2230 IgG, IgA, TNF inflammation,U.S. Pat. No. IgM, or receptor autoimmune 5,808,029, IgE disordersissued (excluding Sep. 15, the first 1998 domain) IgG1 CD4 AIDS Capon etal. receptor (1989), Nature 337: 525-531 IgG1, N-terminus anti-cancer,Harvill et al. IgG3 of IL-2 antiviral (1995), Immunotech., 1: 95-105IgG1 C-terminus osteoarthritis; WO 97/23614, of OPG bone densitypublished Jul. 3, 1997 IgG1 N-terminus anti-obesity PCT/US of leptin97/23183, filed Dec. 11, 1997 Human IgCγ1 CTLA-4 autoimmune Linsley(1991), disorders J. Exp. Med., 174: 561-569

In one example, all or a portion of the human IgG hinge, CH2 and CH3regions may be fused at either the N-terminus or C-terminus of a β10polypeptide of this invention using methods known to the skilledartisan. The resulting β10-fusion polypeptide or α2/β10-fusionpolypeptide may be purified by use of a Protein A affinity column.Peptides and proteins fused to an Fc region have been found to exhibit asubstantially greater half-life in vivo than the unfused counterpart.Also, a fusion to an Fc region allows for dimerization/multimerizationof the fusion polypeptide. The Fc region may be a naturally occurring Fcregion, or may be altered to improve certain qualities, such astherapeutic qualities, circulation time, reduce aggregation, etc.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J.Applied Math., 48:1073 (1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., supra). The well known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast fifty (50) contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., Atlas ofProtein Sequence and Structure, vol. 5, supp. 3 (1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA,89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also usedby the algorithm.

Preferred parameters for a polypeptide sequence comparison include thefollowing:

-   -   Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);    -   Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl.        Acad. Sci. USA, 89:10915-10919 (1992);    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisonsinclude the following:

-   -   Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);    -   Comparison matrix: matches=+10, mismatch=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3        The GAP program is also useful with the above parameters. The        aforementioned parameters are the default parameters for nucleic        acid molecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA to DNA, protein to protein,protein to DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

Synthesis

It will be appreciated by those skilled in the art the nucleic acid andpolypeptide molecules described herein may be produced by recombinantand other means.

Nucleic Acid Molecules

The nucleic acid molecules encode a polypeptide comprising the aminoacid sequence of a β10 polypeptide of this invention can readily beobtained in a variety of ways including, without limitation, chemicalsynthesis, cDNA or genomic library screening, expression libraryscreening and/or PCR amplification of cDNA.

Recombinant DNA methods used herein are generally those set forth inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and/or Ausubelet al., eds., Current Protocols in Molecular Biology, Green PublishersInc. and Wiley and Sons, NY (1994). The present invention provides fornucleic acid molecules as described herein and methods for obtaining themolecules.

Where a gene encoding the amino acid sequence of a β10 polypeptide hasbeen identified from one species, all or a portion of that gene may beused as a probe to identify orthologs or related genes from the samespecies. The probes or primers may be used to screen cDNA libraries fromvarious tissue sources believed to express the β10 polypeptide. Inaddition, part or all of a nucleic acid molecule having the sequence setforth in SEQ ID NO: 2 may be used to screen a genomic library toidentify and isolate a gene encoding the amino acid sequence of a β10polypeptide. Typically, conditions of moderate or high stringency willbe employed for screening to minimize the number of false positivesobtained from the screen.

Nucleic acid molecules encoding the amino acid sequence of a β10polypeptide may also be identified by expression cloning which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingof an antibody or other binding partner (e.g., receptor or ligand) tocloned proteins which are expressed and displayed on a host cellsurface. The antibody or binding partner is modified with a detectablelabel to identify those cells expressing the desired clone.

Recombinant expression techniques conducted in accordance with thedescriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence which encodes the amino acid sequenceof a β10 polypeptide into an appropriate vector, one skilled in the artcan readily produce large quantities of the desired nucleotide sequence.The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of a β10 polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded β10 polypeptide may be produced in largeamounts.

Another method for obtaining a suitable nucleic acid sequence is thepolymerase chain reaction (PCR). In this method, cDNA is prepared frompoly(A)+RNA or total RNA using the enzyme reverse transcriptase. Twoprimers, typically complementary to two separate regions of cDNA(oligonucleotides) encoding the amino acid sequence of a β10polypeptide, are then added to the cDNA along with a polymerase such asTaq polymerase, and the polymerase amplifies the cDNA region between thetwo primers.

Another means of preparing a nucleic acid molecule encoding the aminoacid sequence of a β10 polypeptide is chemical synthesis using methodswell known to the skilled artisan such as those described by Engels etal., Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include,inter alia, the phosphotriester, phosphoramidite, and H-phosphonatemethods for nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry. Typically, the DNA encoding the amino acid sequence of a β10polypeptide will be several hundred nucleotides in length. Nucleic acidslarger than about 100 nucleotides can be synthesized as severalfragments using these methods. The fragments can then be ligatedtogether to form the full length nucleotide sequence of a β10polypeptide. Usually, the DNA fragment encoding the amino terminus ofthe polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of the β10polypeptide, depending on whether the polypeptide produced in the hostcell is designed to be secreted from that cell. Other methods known tothe skilled artisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for the optimal expression of β10 polypeptide or α2/β10heterodimer in a given host cell. Particular codon alterations willdepend upon the β10 polypeptide(s) and host cell(s) selected forexpression. Such “codon optimization” can be carried out by a variety ofmethods, for example, by selecting codons which are preferred for use inhighly expressed genes in a given host cell. Computer algorithms whichincorporate codon frequency tables such as “Ecohigh.cod” for codonpreference of highly expressed bacterial genes may be used and areprovided by the University of Wisconsin Package Version 9.0, GeneticsComputer Group, Madison, Wis. Other useful codon frequency tablesinclude “Celegans_high.cod”, “Celegans_low.cod”, “Drosophila_high.cod”,“Human_high.cod”, “Maize_high.cod”, and “Yeast_high.cod”.

Vectors and Host Cells

When contemplating expression of an α2/β10 heterodimer, it should beunderstood that the β10 polypeptide expression vector as well as anexpression vector encoding α2 polypeptide can both be introduced (forexample, transformed, co-transformed, transfected, co-transfected,transduced, co-transduced) into a host cell, cell line, tissue, organ,animal or plant. It is also understood that introduction of a β10polypeptide expression vector alone into a host cell, cell line, tissue,organ, animal or plant that already produces α2 polypeptide can resultin de novo or enhanced production of an α2/β10 heterodimer. As describedabove in the heterodimerization assay recombinant polyHis and FLAGtagged α2/β10 heterodimer was produced by co-transfection of mammaliancells. Heterodimeric glycoprotein hormones such as CG can also beassembled in vitro upon co-incubation of, for example, isolatedalpha-subunit and isolated CG-β subunit under suitable conditions [seeBlithe and Iles, Endocrinology, volume 136, pages 903-910 (1995)].α2/β10 heterodimer could similarly be assembled in vitro upon incubationof isolated α2 polypeptide and isolated β10 polypeptide. The resultmight be a mixture of α2 polypeptide, β10 polypeptide and α2/β10heterodimer. Each of these products could be isolated in purified formusing conventional methods such as size exclusion chromatography and/orimmunoaffinity chromatography. In this regard α2 polypeptide alone andβ10 polypeptide alone can be separately produced and secreted frommammalian cells as described below. A human α2-polyHis-tag mammalianexpression vector was transfected into 293 cells and serum freeconditioned media was harvested after 72 hours. Western blot of thisconditioned media was probed with affinity purified anti-α2 rabbitpolyclonal antibodies (example 4) that had been conjugated to HorseRadish Peroxidase (Linx HRP Rapid Protein Conjugation Kit, cat#K8050-01, Invitrogen Corp., Carlsbad, Calif.). A strong band wasobserved using the ECL Western Blot detection kit (cat#RPN 2106,Amersham Pharmacia Biotech, Piscataway, N.J.) demonstrating that a humanα2 polypeptide could be secreted in the absence of β10 polypeptide. Ahuman β10-FLAG-tag mammalian expression vector was transfected into 293cells and serum free conditioned media was harvested after 72 hours.Western blot of this conditioned media was probed with a biotinylatedanti-FLAG M2 monoclonal antibody (cat# F9291, Sigma, St. Louis, Mo.) andthen probed with Streptavidin linked Horse Radish Peroxidase (RPN 1231,Amersham Life Sciences). A strong band was observed using the ECLWestern Blot detection kit (cat#RPN 2106, Amersham Pharmacia Biotech,Piscataway, N.J.) demonstrating that a human β10 polypeptide could besecreted in the absence of α2 polypeptide.

A nucleic acid molecule encoding the amino acid sequence of a β10polypeptide may be inserted into an appropriate expression vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of a β10 polypeptide according to thisinvention may be amplified/expressed in prokaryotic, yeast, insect(baculovirus systems), and/or eukaryotic host cells. Selection of thehost cell will depend in part on whether the β10 polypeptide or α2/β10heterodimer is to be post-translationally modified (e.g., glycosylatedand/or phosphorylated). If so, yeast, insect, or mammalian host cellsare preferable. For a review of expression vectors, see Meth. Enz., v.185, D. V. Goeddel, ed. Academic Press Inc., San Diego, Calif. (1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the β10polypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or other “tag” such as FLAG, HA (hemaglutininInfluenza virus) or myc for which commercially available antibodiesexist. This tag is typically fused to the polypeptide upon expression ofthe polypeptide, and can serve as a means for affinity purification ofthe β10 polypeptide or α2/β10 heterodimer from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified β10 polypeptide orα2/β10 heterodimer by various means such as using certain peptidases forcleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source) or synthetic, or theflanking sequences may be native sequences which normally function toregulate β10 polypeptide expression. As such, the source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequence is functional in, and can be activated by, the host cellmachinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein other than the β10 gene flankingsequences will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species. Where the flanking sequence is not known, a fragment ofDNA containing a flanking sequence may be isolated from a larger pieceof DNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion to produce the proper DNA fragment followed by isolation usingagarose gel purification, Qiagen® column chromatography (Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of β10 polypeptide or α2/β10 heterodimer. If thevector of choice does not contain an origin of replication site, one maybe chemically synthesized based on a known sequence, and ligated intothe vector. For example, the origin of replication from the plasmidpBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass.) issuitable for most Gram-negative bacteria and various origins (e.g.,SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) orpapillomaviruses such as HPV or BPV) are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (for example, the SV40 origin isoften used only because it contains the early promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene which will beexpressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes a β10 polypeptide of this invention. As a result,increased quantities of the β10 polypeptide or α2/β10 heterodimer aresynthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the β10 polypeptide to beexpressed. The Shine-Dalgarno sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth herein and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct the β10 polypeptideor α2/β10 heterodimer out of the host cell. Typically, a nucleotidesequence encoding the signal sequence is positioned in the coding regionof a β10 nucleic acid molecule, or directly at the 5′ end of a β10polypeptide coding region. Many signal sequences have been identified,and any of those that are functional in the selected host cell may beused in conjunction with a β10 nucleic acid molecule. Therefore, asignal sequence may be homologous (naturally occurring) or heterologousto a β10 gene or cDNA. Additionally, a signal sequence may be chemicallysynthesized using methods described herein. In most cases, the secretionof a β10 polypeptide or α2/β10 heterodimer from the host cell via thepresence of a signal peptide will result in the removal of the signalpeptide from the secreted β10 polypeptide or α2/β10 heterodimer. Thesignal sequence may be a component of the vector, or it may be a part ofa β10 nucleic acid molecule that is inserted into the vector.

Included within the scope of this invention is the use of either anucleotide sequence encoding a native β10 polypeptide signal sequencejoined to a β10 polypeptide coding region or a nucleotide sequenceencoding a heterologous signal sequence joined to an β10 polypeptidecoding region. The heterologous signal sequence selected should be onethat is recognized and processed, i.e., cleaved by a signal peptidase,by the host cell. For prokaryotic host cells that do not recognize andprocess the native β10 polypeptide signal sequence, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, orheat-stable enterotoxin II leaders. For yeast secretion, the native β10polypeptide signal sequence may be substituted by the yeast invertase,alpha factor, or acid phosphatase leaders. In mammalian cell expressionthe native signal sequence is satisfactory, although other mammaliansignal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, addpresequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the N-terminus. Alternatively,use of some enzyme cleavage sites may result in a slightly truncatedform of the desired β10 polypeptide or α2/β10 heterodimer if the enzymecuts at such area within the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the β10 gene, especially where the gene used is a fulllength genomic sequence or a fragment thereof. Where the intron is notnaturally occurring within the gene (as for most cDNAs), the intron(s)may be obtained from another source. The position of the intron withrespect to flanking sequences and the β10 gene is generally important,as the intron must be transcribed to be effective. Thus, when a β10 cDNAmolecule is being transcribed, the preferred position for the intron is3′ to the transcription start site, and 5′ to the polyA transcriptiontermination sequence. Preferably, the intron or introns will be locatedon one side or the other (i.e., 5′ or 3′) of the cDNA such that it doesnot interrupt the coding sequence. Any intron from any source, includingany viral, prokaryotic and eukaryotic (plant or animal) organisms, maybe used to practice this invention, provided that it is compatible withthe host cell(s) into which it is inserted. Also included herein aresynthetic introns. Optionally, more than one intron may be used in thevector.

The expression and cloning vectors of the present invention will eachtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding a β10 polypeptide. Promotersare untranscribed sequences located upstream (5′) to the start codon ofa structural gene (generally within about 100 to 1000 base pairs) thatcontrol the transcription of the structural gene. Promoters areconventionally grouped into one of two classes, inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding a β10 polypeptide byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.The native β10 gene promoter sequence may be used to directamplification and/or expression of a β10 nucleic acid molecule. Aheterologous promoter is preferred, however, if it permits greatertranscription and higher yields of the expressed protein as compared tothe native promoter, and if it is compatible with the host cell systemthat has been selected for use.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase, atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirsequences have been published, thereby enabling one skilled in the artto ligate them to the desired DNA sequence(s), using linkers or adaptersas needed to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40). Other suitable mammalian promotersinclude heterologous mammalian promoters, e.g., heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest in controlling β10 genetranscription include, but are not limited to: the SV40 early promoterregion (Bernoist Chambon, Nature, 290:304-310, 1981); the CMV promoter;the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797, 1980); the herpes thymidinekinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA, 78:144-1445,1981); the regulatory sequences of the metallothionine gene (Brinster etal., Nature, 296:39-42, 1982); prokaryotic expression vectors such asthe beta-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad.Sci. USA, 75:3727-3731, 1978); or the tac promoter (DeBoer, et al.,Proc. Natl. Acad. Sci. USA, 80:21-25, 1983). Also of interest are thefollowing animal transcriptional control regions, which exhibit tissuespecificity and have been utilized in transgenic animals: the elastase Igene control region which is active in pancreatic acinar cells (Swift etal., Cell, 38:639-646, 1984; Ornitz et al., Cold Spring Harbor Symp.Quant. Biol., 50:399-409 (1986); MacDonald, Hepatology, 7:425-515,1987); the insulin gene control region which is active in pancreaticbeta cells (Hanahan, Nature, 315:115-122, 1985); the immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,Cell, 38:647-658 (1984); Adames et al., Nature, 318:533-538 (1985);Alexander et al., Mol. Cell. Biol., 7:1436-1444, 1987); the mousemammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., Cell, 45:485-495, 1986);the albumin gene control region which is active in liver (Pinkert etal., Genes and Devel., 1:268-276, 1987); the alphafetoprotein genecontrol region which is active in liver (Krumlauf et al., Mol. Cell.Biol., 5:1639-1648, 1985; Hammer et al., Science, 235:53-58, 1987); thealpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., Genes and Devel., 1:161-171, 1987); the beta-globin genecontrol region which is active in myeloid cells (Mogram et al., Nature,315:338-340, 1985; Kollias et al., Cell, 46:89-94, 1986); the myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., Cell, 48:703-712, 1987); the myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, Nature, 314:283-286, 1985); and the gonadotropic releasinghormone gene control region which is active in the hypothalamus (Masonet al., Science, 234:1372-1378, 1986).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding a β10 polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation of eukaryotic promoters. While an enhancer may be splicedinto the vector at a position 5′ or 3′ to a β10 nucleic acid molecule,it is typically located at a site 5′ from the promoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe desired flanking sequences are not already present in the vector,they may be individually obtained and ligated into the vector. Methodsused for obtaining each of the flanking sequences are well known to oneskilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, Carlsbad, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15□ (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. WO90/14363) andpFastBacDual (Gibco/BRL, Grand Island, N.Y.).

Additional suitable vectors include, but are not limited to, cosmids,plasmids or modified viruses, but it will be appreciated that the vectorsystem must be compatible with the selected host cell. Such vectorsinclude, but are not limited to plasmids such as Bluescript® plasmidderivatives (a high copy number ColE1-based phagemid, Stratagene CloningSystems Inc., La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian,yeast, or virus vectors such as a baculovirus expression system (pBacPAKplasmid derivatives, Clontech, Palo Alto, Calif.).

After the vector has been constructed and a nucleic acid moleculeencoding a β10 polypeptide has been inserted into the proper site of thevector, the completed vector may be inserted into a suitable host cellfor amplification and/or polypeptide expression. The transformation ofan expression vector for a β10 polypeptide into a selected host cell maybe accomplished by well known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection or the DEAE-dextran method or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., supra.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell or a vertebrate cell).The host cell, when cultured under appropriate conditions, synthesizes aβ10 polypeptide or α2/β10 heterodimer which can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted). The selection of an appropriate host cell will depend uponvarious factors, such as desired expression levels, polypeptidemodifications that are desirable or necessary for activity, such asglycosylation or phosphorylation, and ease of folding into abiologically active molecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209. Examples include, butare not limited to, mammalian cells, such as Chinese hamster ovary cells(CHO) (ATCC No. CCL61) CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad.Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293Tcells (ATCC No. CRL1573), or 3T3 cells (ATCC No. CCL92). The selectionof suitable mammalian host cells and methods for transformation,culture, amplification, screening and product production andpurification are known in the art. Other suitable mammalian cell lines,are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No.CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines, which are available from the ATCC. Each of these cell lines isknown by and available to those skilled in the art of proteinexpression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, (ATCC No. 33694) DH5α, DH10, and MC1061 (ATCC No. 53338)) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomycesspp., and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for the expression of the β10 polypeptides orα2/β10 heterodimers of the present invention. Preferred yeast cellsinclude, for example, Saccharomyces cerivisae and Pichia pastoris.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin.Biotechnol., 4:564-572 (1993); and Lucklow et al. (J. Virol.,67:4566-4579 (1993). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

One may also use transgenic animals to express glycosylated β10polypeptide or α2/β10 heterodimer. For example, one may use a transgenicmilk-producing animal (a cow or goat, for example) and obtainglycosylated β10 polypeptide or α2/β10 heterodimer in the animal milk.One may also use plants to produce β10 polypeptide or α2/β10 heterodimerhowever, in general, the glycosylation occurring in plants is differentfrom that produced in mammalian cells, and may result in a glycosylatedproduct which is not suitable for human therapeutic use.

Polypeptide Production

Host cells comprising a β10 polypeptide or α2/β10 heterodimer expressionvector may be cultured using standard media well known to the skilledartisan. The media will usually contain all nutrients necessary for thegrowth and survival of the cells. Suitable media for culturing E. colicells include, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells include Roswell ParkMemorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium(MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which maybe supplemented with serum and/or growth factors as indicated by theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate and/or fetal calf serum, as necessary.

Typically, an antibiotic or other compound useful for selective growthof transformed cells is added as a supplement to the media. The compoundto be used will be dictated by the selectable marker element present onthe plasmid with which the host cell was transformed. For example, wherethe selectable marker element is kanamycin resistance, the compoundadded to the culture medium will be kanamycin. Other compounds forselective growth include ampicillin, tetracycline, and neomycin.

The amount of β10 polypeptide or α2/β10 heterodimer produced by a hostcell can be evaluated using standard methods known in the art. Suchmethods include, without limitation, Western blot analysis,SDS-polyacrylamide gel electrophoresis, non-denaturing gelelectrophoresis, HPLC separation, immunoprecipitation, and/or activityassays such as DNA binding gel shift assays.

If a β10 polypeptide or α2/β10 heterodimer has been designed to besecreted from the host cells, the majority of β10 polypeptide or α2/β10heterodimer may be found in the cell culture medium. If however, the β10polypeptide or α2/β10 heterodimer is not secreted from the host cells,it will be present in the cytoplasm and/or the nucleus (for eukaryotichost cells) or in the cytosol (for bacterial host cells).

For β10 polypeptide or α2/β10 heterodimer situated in the host cellcytoplasm and/or the nucleus (for eukaryotic host cells) or in thecytosol (for bacterial host cells), intracellular material (includinginclusion bodies for gram-negative bacteria) can be extracted from thehost cell using any standard technique known to the skilled artisan. Forexample, the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

If the β10 polypeptide or α2/β10 heterodimer has formed inclusion bodiesin the cytosol, the inclusion bodies can often bind to the inner and/orouter cellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedat pH extremes or with a chaotropic agent such as a detergent,guanidine, guanidine derivatives, urea, or urea derivatives in thepresence of a reducing agent such as dithiothreitol at alkaline pH ortris carboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The β10 polypeptide or α2/β10heterodimer in its now soluble form can then be analyzed using gelelectrophoresis, immuno-precipitation or the like. If it is desired toisolate the β10 polypeptide or α2/β10 heterodimer, isolation may beaccomplished using standard methods such as those described herein andin Marston et al., Meth. Enz., 182:264-275 (1990).

In some cases, the β10 polypeptide or α2/β10 heterodimer may not bebiologically active upon isolation. Various methods for “refolding” orconverting the β10 polypeptide or α2/β10 heterodimer to its tertiarystructure and generating disulfide linkages can be used to restorebiological activity. Such methods include exposing the solubilized β10polypeptide or α2/β10 heterodimer to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol (DTT)/dithiane DTT, and 2-2mercaptoethanol (bME)/dithio-b(ME). A cosolvent may be used to increase the efficiency of therefolding, and the more common reagents used for this purpose includeglycerol, polyethylene glycol of various molecular weights, arginine andthe like.

If inclusion bodies are not formed to a significant degree uponexpression of β10 polypeptide or α2/β10 heterodimer then the β10polypeptide or α2/β10 heterodimer will be found primarily in thesupernatant after centrifugation of the cell homogenate. The β10polypeptide or α2/β10 heterodimer may be further isolated from thesupernatant using methods such as those described herein.

The purification of β10 polypeptide or α2/β10 heterodimer from solutioncan be accomplished using a variety of techniques. If the β10polypeptide or α2/β10 heterodimer has been synthesized such that itcontains a tag such as Hexahistidine (β10 polypeptide-hexaHis,α2/β10-hexaHis heterodimer) or other small peptide such as FLAG (EastmanKodak Co., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) ateither its carboxyl or amino terminus, it may be purified in a one-stepprocess by passing the solution through an affinity column where thecolumn matrix has a high affinity for the tag.

For example, polyhistidine binds with great affinity and specificity tonickel, thus an affinity column of nickel (such as the Qiagen® nickelcolumns) can be used for purification of β10 polypeptide-polyHis orα2/β10-hexaHis heterodimer. See for example, Ausubel et al., eds.,Current Protocols in Molecular Biology, Section 10.11.8, John Wiley &Sons, New York (1993).

Additionally, the β10 polypeptide or α2/β10 heterodimer may be purifiedthrough the use of a monoclonal antibody which is capable ofspecifically recognizing and binding to the β10 polypeptide or α2/β10heterodimer.

Suitable procedures for purification thus include, without limitation,affinity chromatography, immunoaffinity chromatography, ion exchangechromatography, molecular sieve chromatography, High Performance LiquidChromatography (HPLC), electrophoresis (including native gelelectrophoresis) followed by gel elution, and preparative isoelectricfocusing (“Isoprime” machine/technique, Hoefer Scientific, SanFrancisco, Calif.). In some cases, two or more purification techniquesmay be combined to achieve increased purity.

β10 polypeptides or α2/β10 heterodimers may also be prepared by chemicalsynthesis methods (such as solid phase peptide synthesis) usingtechniques known in the art, such as those set forth by Merrifield etal., J. Am. Chem. Soc., 85:2149 (1963), Houghten et al., Proc Natl Acad.Sci. USA, 82:5132 (1985), and Stewart and Young, Solid Phase PeptideSynthesis, Pierce Chemical Co., Rockford, Ill. (1984). Such β10polypeptides or α2/β10 heterodimers may be synthesized with or without amethionine on the amino terminus. Chemically synthesized β10polypeptides or α2/β10 heterodimers may be oxidized using methods setforth in these references to form disulfide bridges. Chemicallysynthesized β10 polypeptides or α2/β10 heterodimers are expected to havecomparable biological activity to the corresponding β10 polypeptides(when heterodimerized to α2) or α2/β10 heterodimers producedrecombinantly or purified from natural sources, and thus may be usedinterchangeably with a recombinant or natural β10 polypeptide or α2/β10heterodimer.

Another means of obtaining a β10 polypeptide or α2/β10 heterodimeraccording to this invention is via purification from biological samplessuch as source tissues and/or fluids in which the β10 polypeptide orα2/β10 heterodimer is naturally found. Such purification can beconducted using methods for protein purification as described herein.The presence of the β10 polypeptide or α2/β10 heterodimer duringpurification may be monitored using, for example, a correspondingantibody prepared against recombinantly produced β10 polypeptide orpeptide fragment thereof, or prepared against recombinantly producedα2/β10 heterodimer or peptide fragment.

A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and can be used to producepolypeptides having specificity for β10 polypeptides or α2/β10heterodimers of this invention. See for example, Roberts, et al., Proc.Natl. Acad. Sci., 94:12297-12303 (1997), which describes the productionof fusion proteins between an mRNA and its encoded peptide. See alsoU.S. Pat. No. 5,824,469, which describes methods of obtainingoligonucleotides capable of carrying out a specific biological function.The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those which exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

U.S. Pat. Nos. 5,763,192, 5,814,476, 5,723,323, and 5,817,483 describeprocesses for producing peptides or polypeptides. This is done byproducing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

Chemical Derivatives

Chemically modified derivatives of the β10 polypeptides or α2/β10heterodimers of this invention may be prepared by one skilled in theart, given the disclosures set forth hereinbelow. Such β10 polypeptideor α2/β10 heterodimer derivatives are modified in a manner that isdifferent, either in the type or location of the molecules naturallyattached to the β10 polypeptide or α2/β10 heterodimer. Derivatives mayinclude molecules formed by the deletion of one or morenaturally-attached chemical groups. The polypeptide comprising the aminoacid sequence of SEQ ID NO: 3, a β10-like polypeptide variant thereof ora α2/β10 heterodimer may be modified by the covalent attachment of oneor more polymers. For example, the polymer selected is typically watersoluble so that the protein to which it is attached does not precipitatein an aqueous environment, such as a physiological environment. Includedwithin the scope of suitable polymers is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer preferably is between about 5 kDa andabout 50 kDa, more preferably between about 12 kDa and about 40 kDa andmost preferably between about 20 kDa and about 35 kDa.

Suitable water soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C₁-C₁₀) alkoxy- oraryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran(such as low molecular weight dextran, of, for example about 6 kD),cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached multimers of the polypeptide comprising theamino acid sequence of SEQ ID NO: 3, a polypeptide variant thereof or aα2/β10 heterodimer.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides orheterodimers will generally comprise the steps of (a) reacting thepolypeptide or heterodimer with the activated polymer molecule (such asa reactive ester or aldehyde derivative of the polymer molecule) underconditions whereby the polypeptide comprising the amino acid sequence ofSEQ ID NO: 3, or a polypeptide variant thereof or an α2/β10 heterodimerbecomes attached to one or more polymer molecules, and (b) obtaining thereaction product(s). The optimal reaction conditions will be determinedbased on known parameters and the desired result. For example, thelarger the ratio of polymer molecules:protein, the greater thepercentage of attached polymer molecule. In one embodiment, the β10polypeptide or α2/β10 heterodimer derivative may have a single polymermolecule moiety at the amino terminus. See, for example, U.S. Pat. No.5,234,784.

The pegylation of the polypeptide or heterodimer specifically may becarried out by any of the pegylation reactions known in the art, asdescribed for example in the following references: Francis et al., Focuson Growth Factors, 3:4-10 (1992); EP 0154316; EP 0401384 and U.S. Pat.No. 4,179,337. For example, pegylation may be carried out via anacylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer) as described herein. For the acylation reactions, thepolymer(s) selected should have a single reactive ester group. Forreductive alkylation, the polymer(s) selected should have a singlereactive aldehyde group. A reactive aldehyde is, for example,polyethylene glycol propionaldehyde, which is water stable, or monoC₁-C₁₀ alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.5,252,714).

In another embodiment, a β10 polypeptide or α2/β10 heterodimer may bechemically coupled to biotin, and the biotin-β10 polypeptide orbiotin-α2/β10 heterodimer molecules which are conjugated are thenallowed to bind to avidin, resulting in tetravalent avidin/biotin/β10polypeptide molecules or avidin/biotin/α2/β10 heterodimer molecules. β10polypeptides or α2/β10 heterodimers may also be covalently coupled todinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugatesprecipitated with anti-DNP or anti-TNP-IgM to form decameric conjugateswith a valency of 10.

Generally, conditions which may be alleviated or modulated by theadministration of the present β10 polypeptide or α2/β10 heterodimerderivatives include those described herein for β10 polypeptides orα2/β10 heterodimers. However, the β10 polypeptide or α2/β10 heterodimerderivatives disclosed herein may have additional activities, enhanced orreduced biological activity, or other characteristics, such as increasedor decreased half-life, as compared to the non-derivatized molecules.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents, rabbits, goats,or sheep, or other farm animals, in which the gene (or genes) encodingthe native β10 polypeptide has (have) been disrupted (“knocked out”)such that the level of expression of this gene or genes is (are)significantly decreased or completely abolished. Such animals may beprepared using techniques and methods such as those described in U.S.Pat. No. 5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents, rabbits, goats, sheep, or other farm animals, inwhich either the native form of the β10 gene(s) for that animal or aheterologous β10 gene(s) is (are) over-expressed by the animal, therebycreating a “transgenic” animal. Such transgenic animals may be preparedusing well known methods such as those described in U.S. Pat. No.5,489,743 and PCT application No. WO94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the β10 polypeptides is/are either activatedor inactivated (e.g., by using homologous recombination methods) toalter the level of expression of native β10 polypeptide.

These non-human animals may be used for drug candidate screening. Insuch screening, the impact of a drug candidate on the animal may bemeasured. For example, drug candidates may decrease or increase theexpression of the β10 gene. In certain embodiments, the amount of β10polypeptide that is produced may be measured after the exposure of theanimal to the drug candidate. Additionally, in certain embodiments, onemay detect the actual impact of the drug candidate on the animal. Forexample, the overexpression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

Microarray

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array has numerous copies of asingle species of DNA which acts as a target for hybridization for itscognate mRNA. In expression profiling using DNA microarray technology,mRNA is first extracted from a cell or tissue sample and then convertedenzymatically to fluorescently labeled cDNA. This material is hybridizedto the microarray and unbound cDNA is removed by washing. The expressionof discrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA which is specifically bound toeach target DNA. In this way, the expression of thousands of genes canbe quantitated in a high throughput, parallel manner from a singlesample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the β10 molecules of the invention,including, but not limited to: the identification and validation of β10disease-related genes as targets for therapeutics; molecular toxicologyof β10 molecules and inhibitors thereof; stratification of populationsand generation of surrogate markers for clinical trials; and enhancingβ10 related small molecule drug discovery by aiding in theidentification of selective compounds in high throughput screens (HTS).

Selective Binding Agents

As used herein, the term “selective binding agent” refers to a moleculewhich has specificity for one or more β10 polypeptides or α2/β10heterodimers. Suitable selective binding agents include, but are notlimited to, antibodies and derivatives thereof, polypeptides, and smallmolecules. Suitable selective binding agents may be prepared usingmethods known in the art. An exemplary β10 polypeptide or α2/β10heterodimer selective binding agent of the present invention is capableof binding a certain portion of the β10 polypeptide or α2/β10heterodimer thereby inhibiting the binding of the β10 polypeptide orα2/β10 heterodimer to the β10 polypeptide or α2/β10 heterodimerreceptor(s).

Selective binding agents such as antibodies and antibody fragments thatbind β10 polypeptides or α2/β10 heterodimers are within the scope of thepresent invention. The antibodies may be polyclonal includingmonospecific polyclonal, monoclonal (MAbs), recombinant, chimeric,humanized such as CDR-grafted, human, single chain, and/or bispecific,as well as fragments, variants or derivatives thereof. Antibodyfragments include those portions of the antibody which bind to anepitope on the β10 polypeptide or α2/β10 heterodimer. Examples of suchfragments include Fab and F(ab′) fragments generated by enzymaticcleavage of full-length antibodies. Other binding fragments includethose generated by recombinant DNA techniques, such as the expression ofrecombinant plasmids containing nucleic acid sequences encoding antibodyvariable regions.

Polyclonal antibodies directed toward β10 polypeptide or α2/β10heterodimer generally are produced in animals (e.g., rabbits or mice) bymeans of multiple subcutaneous or intraperitoneal injections of β10polypeptide or α2/β10 heterodimer and an adjuvant. It may be useful toconjugate a β10 polypeptide or α2/β10 heterodimer to a carrier proteinthat is immunogenic in the species to be immunized, such as keyholelimpet heocyanin, serum, albumin, bovine thyroglobulin, or soybeantrypsin inhibitor. Also, aggregating agents such as alum are used toenhance the immune response. After immunization, the animals are bledand the serum is assayed for anti-β10 polypeptide or anti-α2/β10heterodimer antibody titer.

Monoclonal antibodies directed toward β10 polypeptide or α2/β10heterodimer are produced using any method which provides for theproduction of antibody molecules by continuous cell lines in culture.Examples of suitable methods for preparing monoclonal antibodies includethe hybridoma methods of Kohler et al., Nature, 256:495-497 (1975) andthe human B-cell hybridoma method, Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). Alsoprovided by the invention are hybridoma cell lines which producemonoclonal antibodies reactive with β10 polypeptide or α2/β10heterodimer of this invention.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy and/or light chain is identical with or homologous to acorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity. See, U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci., 81:6851-6855 (1985).

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. See U.S. Pat. Nos. 5,585,089, and 5,693,762.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. Humanization can beperformed, for example, using methods described in the art (Jones etal., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substitutingat least a portion of a rodent complementarity-determining region (CDR)for the corresponding regions of a human antibody.

Also encompassed by the invention are human antibodies which bind β10polypeptide or α2/β10 heterodimer. Using transgenic animals (e.g., mice)that are capable of producing a repertoire of human antibodies in theabsence of endogenous immunoglobulin production such antibodies areproduced by immunization with a β10 polypeptide or α2/β10 heterodimerantigen (i.e., having at least 6 contiguous amino acids), optionallyconjugated to a carrier. See, for example, Jakobovits et al., Proc.Natl. Acad. Sci., 90:2551-2555 (1993); Jakobovits et al., Nature362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993). Inone method, such transgenic animals are produced by incapacitating theendogenous loci encoding the heavy and light immunoglobulin chainstherein, and inserting loci encoding human heavy and light chainproteins into the genome thereof. Partially modified animals, that isthose having less than the full complement of modifications, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies with human (rather than e.g., murine) amino acidsequences, including variable regions which are immunospecific for theseantigens. See PCT application nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCTapplication nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP546073A1. Human antibodies may also be produced by the expression ofrecombinant DNA in host cells or by expression in hybridoma cells asdescribed herein.

In an alternative embodiment, human antibodies can be produced fromphage-display libraries (Hoogenboom et al., J. Mol. Biol. 227:381(1991); Marks et al., J. Mol. Biol. 222:581 (1991). These processesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in PCT Application no. PCT/US98/17364, which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Monoclonal (e.g., human)antibodies may be produced by the expression of recombinant DNA in hostcells or by expression in hybridoma cells as described herein.

The anti-β10 polypeptide antibodies or anti-α2/β10 heterodimerantibodies of the invention may be employed in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158, CRC Press, Inc., 1987) for the detection andquantitation of β10 polypeptide or α2/β10 heterodimer. The antibodieswill bind the β10 polypeptide or α2/β10 heterodimer with an affinitywhich is appropriate for the assay method being employed.

For diagnostic applications, in certain embodiments, anti-β10polypeptide antibodies or anti-α2/β10 heterodimer antibodies may belabeled with a detectable moiety. The detectable moiety can be any onewhich is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,α-galactosidase, or horseradish peroxidase (Bayer et al., Meth. Enz.,184:138-163 (1990).

Competitive binding assays rely on the ability of a labeled standard(e.g., a β10 polypeptide or α2/β10 heterodimer or an immunologicallyreactive portion thereof) to compete with the test sample analyte (a β10polypeptide or α2/β10 heterodimer) for binding with a limited amount ofanti-β10 polypeptide antibody or anti-α2/β10 heterodimer antibody. Theamount of β10 polypeptide or α2/β10 heterodimer in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

The selective binding agents, including anti-β10 polypeptide antibodiesor anti-α2/β10 heterodimer antibodies, also are useful for in vivoimaging. An antibody labeled with a detectable moiety may beadministered to an animal, preferably into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.The antibody may be labeled with any moiety that is detectable in ananimal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Selective binding agents of the invention, including antibodies, may beused as therapeutics. These therapeutic agents are generally agonists orantagonists, in that they either enhance or reduce, respectively, atleast one of the biological activities of a β10 polypeptide or α2/β10heterodimer according to this invention. In one embodiment, antagonistantibodies of the invention are antibodies or binding fragments thereofwhich are capable of specifically binding to a β10 polypeptide or α2/β10heterodimer and which are capable of inhibiting or eliminating thefunctional activity of a β10 polypeptide or α2/β10 heterodimer in vivoor in vitro. In preferred embodiments, the selective binding agent,e.g., an antagonist antibody, will inhibit the functional activity of aβ10 polypeptide or α2/β10 heterodimer by at least about 50%, andpreferably by at least about 80%. In another embodiment, the selectivebinding agent may be an antibody that is capable of interacting with aβ10 polypeptide or α2/β10 heterodimer binding partner (a ligand orreceptor) thereby inhibiting or eliminating β10 polypeptide or α2/β10heterodimer activity in vitro or in vivo. Selective binding agents,including agonist and antagonist anti-β10 polypeptide antibodies oranti-α2/β10 heterodimer antibodies, are identified by screening assayswhich are well known in the art.

The invention also relates to a kit comprising β10 polypeptide or α2/β10heterodimer selective binding agents (such as antibodies) and otherreagents useful for detecting β10 polypeptide or α2/β10 heterodimerlevels in biological samples. Such reagents may include, a detectablelabel, blocking serum, positive and negative control samples, anddetection reagents.

β10 polypeptide or α2/β10 heterodimer of this invention can also be usedto clone β10 polypeptide or α2/β10 heterodimer receptor(s), using an“expression cloning” strategy. Radiolabeled (125-Iodine) β10 polypeptideor α2/β10 heterodimer or “affinity/activity-tagged” β10 polypeptide orα2/β10 heterodimer (such as an Fc fusion or an alkaline phosphatasefusion) can be used in binding assays to identify a cell type or cellline or tissue that expresses β10 polypeptide or α2/β10 heterodimerreceptor(s). RNA isolated from such cells or tissues would be convertedto cDNA, cloned into a mammalian expression vector, and transfected intomammalian cells (for example, COS, 293) to create an expression library.Radiolabeled or tagged β10 polypeptide or α2/β10 heterodimer would thenbe used as an affinity ligand to identify and isolate the subset ofcells in this library expressing the β10 polypeptide or α2/β10heterodimer receptor(s) on their surface. DNA would be isolated fromthese cells and transfected into mammalian cells to create a secondaryexpression library in which the fraction of cells expressing β10polypeptide or α2/β10 heterodimer receptor(s) would be many-fold higherthan in the original library. This enrichment process would be repeatediteratively until a single recombinant clone containing a β10polypeptide or α2/β10 heterodimer receptor is isolated. Isolation of theβ10 polypeptide or α2/β10 heterodimer receptor(s) would be very usefulin terms of being able to identify or develop novel agonists andantagonists of the β10 polypeptide or α2/β10 heterodimer signalingpathway(s). Such agonists and antagonists would include soluble β10polypeptide or α2/β10 heterodimer receptor(s), anti-β10polypeptide-receptor(s) antibodies or anti-α2/β10heterodimer(s)-receptor(s) antibodies, small molecules or antisenseoligonucleotides, and they could be used in the diagnosis and/ortreatment of one or more of the diseases/disorders listed below.

Assaying for Other Modulators of β10 Polypeptide or α2/β10 HeterodimerActivity

In some situations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity of a β10polypeptide or α2/β10 heterodimer of this invention. Natural orsynthetic molecules that modulate the β10 polypeptide or α2/β10heterodimer may be identified using one or more screening assays, suchas those described herein. Such molecules may be administered either inan ex vivo manner, or in an in vivo manner by injection, or by oraldelivery, implantation device, or the like.

“Test molecule(s)” refers to the molecule(s) that is/are underevaluation for the ability to modulate (i.e., increase or decrease) theactivity of a β10 polypeptide or α2/β10 heterodimer of this invention.Most commonly, a test molecule will interact directly with thepolypeptide or heterodimer. However, it is also contemplated that a testmolecule may also modulate β10 polypeptide or α2/β10 heterodimeractivity indirectly, such as by affecting β10 gene expression, or bybinding to a β10 polypeptide or α2/β10 heterodimer binding partner(e.g., receptor or ligand). In one embodiment, a test molecule will bindto a β10 polypeptide or α2/β10 heterodimer with an affinity constant ofat least about 10⁻⁶ M, preferably about 10⁻⁸ M, more preferably about10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds which interact with β10 polypeptidesor α2/β10 heterodimers of this invention are encompassed by the presentinvention. In certain embodiments, a β10 polypeptide or α2/β10heterodimer is incubated with a test molecule under conditions whichpermit the interaction of the test molecule with the polypeptide, andthe extent of the interaction can be measured. The test molecule(s) canbe screened in a substantially purified form or in a crude mixture.

In certain embodiments, a β10 polypeptide or α2/β10 heterodimer agonistor antagonist may be a protein, peptide, carbohydrate, lipid, or smallmolecular weight molecule which interacts with the β10 polypeptide orα2/β10 heterodimer to regulate its activity. Molecules which regulateβ10 polypeptide or α2/β10 heterodimer expression include nucleic acidswhich are complementary to nucleic acids encoding a β10 polypeptide ofthis invention, or are complementary to nucleic acids sequences whichdirect, control or influence the expression of the β10 polypeptide orα2/β10 heterodimer and which act as anti-sense regulators of expression.

Once a set of test molecules has been identified as interacting with aβ10 polypeptide or α2/β10 heterodimer the molecules may be furtherevaluated for their ability to increase or decrease β10 polypeptide orα2/β10 heterodimer activity. The measurement of the interaction of testmolecules with β10 polypeptides or α2/β10 heterodimers may be carriedout in several formats, including cell-based binding assays, membranebinding assays, solution-phase assays and immunoassays. In general, testmolecules are incubated with a β10 polypeptide or α2/β10 heterodimer fora specified period of time, and β10 polypeptide or α2/β10 heterodimeractivity is determined by one or more assays for measuring biologicalactivity.

The interaction of test molecules with β10 polypeptides or α2/β10heterodimers according to this invention may also be assayed directlyusing polyclonal or monoclonal antibodies in an immunoassay.Alternatively, modified forms of β10 polypeptides or α2/β10 heterodimerscontaining epitope tags as described herein may be used in immunoassays.

In the event that β10 polypeptides or α2/β10 heterodimers displaybiological activity through an interaction with a binding partner (e.g.,a receptor or a ligand), a variety of in vitro assays may be used tomeasure the binding of a β10 polypeptide or α2/β10 heterodimer to thecorresponding binding partner (such as a selective binding agent,receptor, or ligand). These assays may be used to screen test moleculesfor their ability to increase or decrease the rate and/or the extent ofbinding of a β10 polypeptide or α2/β10 heterodimer to its bindingpartner. In one assay, a β10 polypeptide or α2/β10 heterodimer isimmobilized in the wells of a microtiter plate. Radiolabeled β10polypeptide or α2/β10 heterodimer binding partner (for example,iodinated β10 polypeptide or α2/β10 heterodimer binding partner) and thetest molecule(s) can then be added either one at a time (in eitherorder) or simultaneously to the wells. After incubation, the wells canbe washed and counted, using a scintillation counter, for radioactivityto determine the extent to which the binding partner bound to the β10polypeptide or α2/β10 heterodimer. Typically, the molecules will betested over a range of concentrations, and a series of control wellslacking one or more elements of the test assays can be used for accuracyin the evaluation of the results. An alternative to this method involvesreversing the “positions” of the proteins, i.e., immobilizing β10polypeptide or α2/β10 heterodimer binding partner to the microtiterplate wells, incubating with the test molecule and radiolabeled β10polypeptide or α2/β10 heterodimer and determining the extent of β10polypeptide or α2/β10 heterodimer binding. See, for example, chapter 18,Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley& Sons, New York, N.Y. (1995).

As an alternative to radiolabeling, a β10 polypeptide or α2/β10heterodimer or its respective binding partner may be conjugated tobiotin and the presence of biotinylated protein can then be detectedusing streptavidin linked to an enzyme, such as horseradish peroxidase(HRP) or alkaline phosphatase (AP), that can be detectedcolorometrically, or by fluorescent tagging of streptavidin. An antibodydirected to a β10 polypeptide or α2/β10 heterodimer or to a β10polypeptide or α2/β10 heterodimer binding partner and conjugated tobiotin may also be used and can be detected after incubation withenzyme-linked streptavidin linked to AP or HRP.

A β10 polypeptide or α2/β10 heterodimer or a β10 polypeptide or α2/β10heterodimer binding partner can also be immobilized by attachment toagarose beads, acrylic beads or other types of such inert solid phasesubstrates. The substrate-protein complex can be placed in a solutioncontaining the complementary protein and the test compound. Afterincubation, the beads can be precipitated by centrifugation, and theamount of binding between a β10 polypeptide or α2/β10 heterodimer andits respective binding partner can be assessed using the methodsdescribed herein. Alternatively, the substrate-protein complex can beimmobilized in a column, and the test molecule and complementary proteinare passed through the column. The formation of a complex between a β10polypeptide or α2/β10 heterodimer and its respective binding partner canthen be assessed using any of the techniques set forth herein, i.e.,radiolabeling, antibody binding, or the like.

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases the formation of a complex between a β10polypeptide or α2/β10 heterodimer and a corresponding β10 polypeptide orα2/β10 heterodimer binding partner is a surface plasmon resonancedetector system such as the BIAcore assay system (Pharmacia, Piscataway,N.J.). The BIAcore system may be carried out using the manufacturer'sprotocol. This assay essentially involves the covalent binding of eithera β10 polypeptide, α2/β10 heterodimer, β10 polypeptide binding partneror α2/β10 heterodimer binding partner to a dextran-coated sensor chipwhich is located in a detector. The test compound and the othercomplementary protein can then be injected, either simultaneously orsequentially, into the chamber containing the sensor chip. The amount ofcomplementary protein that binds can be assessed based on the change inmolecular mass which is physically associated with the dextran-coatedside of the sensor chip; the change in molecular mass can be measured bythe detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between β10 polypeptide or α2/β10 heterodimer anda corresponding β10 polypeptide or α2/β10 heterodimer binding partner.In these cases, the assays set forth herein can be readily modified byadding such additional test compound(s) either simultaneous with, orsubsequent to, the first test compound. The remainder of the steps inthe assay are as set forth herein.

In vitro assays such as those described herein may be usedadvantageously to screen large numbers of compounds for effects oncomplex formation by the β10 polypeptide or α2/β10 heterodimer and acorresponding β10 polypeptide or α2/β10 heterodimer binding partner. Theassays may be automated to screen compounds generated in phage display,synthetic peptide, and chemical synthesis libraries.

Compounds which increase or decrease the formation of a complex betweena β10 polypeptide or α2/β10 heterodimer and a corresponding β10polypeptide or α2/β10 heterodimer binding partner may also be screenedin cell culture using cells and cell lines expressing either β10polypeptide or α2/β10 heterodimer and a corresponding β10 polypeptide orα2/β10 heterodimer binding partner. Cells and cell lines may be obtainedfrom any mammal, but preferably will be from human or other primate,canine, or rodent sources. The binding of a β10 polypeptide or α2/β10heterodimer to cells expressing the corresponding β10 polypeptide orα2/β10 heterodimer binding partner at the surface is evaluated in thepresence or absence of test molecules, and the extent of binding may bedetermined by, for example, flow cytometry using a biotinylated antibodyto a β10 polypeptide or α2/β10 heterodimer binding partner. Cell cultureassays can be used advantageously to further evaluate compounds thatscore positive in protein binding assays described herein.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase the expression ofthe β10 gene. In certain embodiments, the amount of β10 polypeptide orα2/β10 heterodimer that is produced may be measured after exposure ofthe cell culture to the drug candidate. In certain embodiments, one maydetect the actual impact of the drug candidate on the cell culture. Forexample, the overexpression of a particular gene may have a particularimpact on the cell culture. In such cases, one may test a drugcandidate's ability to increase or decrease the expression of the geneor its ability to prevent or inhibit a particular impact on the cellculture. In other examples, the production of a particular metabolicproduct such as a fragment of a polypeptide, may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease the production of such ametabolic product in a cell culture.

Therapeutic/Diagnostic Applications of β10 Polypeptides, α2/β10Heterodimers and Nucleic Acids

Biological function is anticipated for a β10 polypeptide or an α2/β10heterodimer similar to that of the glycoprotein hormones FAS, TSH, FSH,LH and CG, which, among other things, are known to act as growth factorsin promoting the development (proliferation, differentiation) ofprolactin producing cells, the thyroid gland and the gonads. Theseglycoproteins also act as endocrine hormones in their role as regulatorsof placental, thyroidal and gonadal function. FAS plays a role instimulating prolactin secretion from decidual cells in the placenta, TSHplays a major role in the regulation of basal metabolism via the thyroidgland, and FSH, LH and CG play critical roles in male female fertility,as well as in pregnancy. As such, a β10 polypeptide or an α2/β10heterodimer may also play roles in the regulation of basal metabolism,the development/function of the gonads, fertility and pregnancy.

As shown in the example further below, β10 polypeptide is expressed inbrain, liver, fetal liver, stomach, pituitary, colon, small intestine,thyroid gland, adrenal gland, pancreas, skin, peripheral bloodleucocytes, spleen, testis and placenta. The fact that β10 is expressedin many of the organs and tissues that make up the endocrine systemsuggests an important role for a β10 polypeptide or an α2/β10heterodimer in the regulation and coordination of one or more endocrinesystem functions. The endocrine system is known to exert major controlover metabolism, physiological responses to stress, and the developmentand function of reproductive organs.

The expression of β10 in pituitary, pancreas, adrenal gland, thyroidgland, stomach, small intestine, colon and liver indicates a possiblerole for a β10 polypeptide or an α2/β10 heterodimer in the commonfunction of these organs or tissues, namely, metabolism andenergy/nutritional homeostasis (i.e., energy balance, basal metabolicrate, digestion, glucose homeostasis, distribution of body fat, generalgrowth).

The expression of β10 in the pituitary and adrenal glands indicates apossible role for a β10 polypeptide or an α2/β10 heterodimer in one ofthe critical functions subserved by these two important organs, namely,the body's ability to cope with a variety of environmental andphysiological stresses (for example, infection, fever, inflammation,fasting, high and low blood pressure, anxiety, shock). Consistent withthese possible functions for a β10 polypeptide or an α2/β10 heterodimeris the expression of β10 in cells and organs known to be importantcomponents of the immune system (peripheral blood leucocytes, spleen,small intestine).

In addition, the expression of β10 in pituitary, testis and placentaindicates a possible role for a β10 polypeptide or an α2/β10 heterodimerin the shared function of these organs, specifically, fertility andpregnancy.

β10 polypeptide or an α2/β10 heterodimer may also act as a growth factorinvolved in the regeneration (proliferation and differentiation) oftissues or specialized cell types present in brain, liver, stomach,pituitary, colon, small intestine, thyroid gland, adrenal gland,pancreas, skin, peripheral blood leucocytes, spleen, testis andplacenta.

Consistent with the three major areas of potential β10 polypeptide or anα2/β10 heterodimer function, i.e., (1) metabolism and energy/nutritionalhomeostasis, (2) physiological responses to stress (including immunesystem function) and (3) fertility and pregnancy, is the fact that α2,which forms a heterodimer with β10, is expressed (see Example 2 below)in many of the same organs/tissues (anterior pituitary, placenta,pancreas, adrenal cortex, intestinal crypts and gall bladder mucosa)that play important roles in these 3 major areas.

Based on the above described potential functions, β10 polypeptide or anα2/β10 heterodimer may be useful for the treatment and/or diagnosis ofmetabolic or energy/nutritional homeostasic disorders. Examples of suchdisorders include, but are not limited to, obesity, wasting syndromes(for example, cancer associated cachexia), myopathies, gastrointestinaldisorders, diabetes, growth failure, hypercholesterolemia,atherosclerosis and aging. Other diseases involving metabolic orenergy/nutritional homeostasic disorders are encompassed within thetherapeutic and diagnostic utilities that are part of the invention.

Based on the above described potential functions, β10 polypeptide or anα2/β10 heterodimer may be useful for the treatment and/or diagnosis ofdisorders related to physiological responses to stress (including immunesystem functions). Examples of such disorders include, but are notlimited to, hypertension, immune system dysfunction (for example,excessive inflammation, autoimmune disease, susceptibility to infectionsuch as AIDS, poor wound healing, psoriasis, asthma, arthritis andallergies), shock, anxiety, and high or low blood pressure. Otherdiseases involving physiological responses to stress, including, but notlimited to, immune system functions, are also encompassed within thetherapeutic and diagnostic utilities that are part of the invention.

Based on the above described potential functions, β10 polypeptide or anα2/β10 heterodimer may be useful for the treatment and/or diagnosis ofdisorders related to pregnancy and/or the development and function ofreproductive organs. Examples of such disorders include, but are notlimited to, infertility, fertility (contraception), impotence,endometriosis, menopause, miscarriage, pre-term labor and delivery.Other diseases involving pregnancy and/or the development and functionof reproductive organs are also encompassed within the therapeutic anddiagnostic utilities that are part of the invention.

Based on the fact that the β10 polypeptide or an α2/β10 heterodimer islikely to have hormone/growth-factor activities, β10 polypeptide or anα2/β10 heterodimer may be useful for the treatment and/or diagnosis ofdisorders that could be treated by increasing cell proliferation and/ordifferentiation. Examples of such disorders include, but are not limitedto, tissue damage/degeneration (such as caused by cancer treatments,infections, autoimmune diseases), aging and wound healing. Otherdiseases that could be treated by increasing cell proliferation and/ordifferentiation are also encompassed within the therapeutic anddiagnostic utilities that are part of the invention.

Based on the fact that the β10 polypeptide or an α2/β10 heterodimer islikely to have hormone/growth-factor activities, β10 polypeptide or anα2/β10 heterodimer may be useful for the treatment and/or diagnosis ofdisorders that could be treated by decreasing cell proliferation and/ordifferentiation. Examples of such disorders include, but are not limitedto, cancers, hyperplasias and hypertrophies. Other diseases that couldbe treated by decreasing cell proliferation and/or differentiation arealso encompassed within the therapeutic and diagnostic utilities thatare part of the invention.

Other diseases caused or mediated by undesirable levels of β10polypeptide or an α2/β10 heterodimer are encompassed within thetherapeutic and diagnostic utilities that are part of the invention. Byway of illustration, such undesirable levels include excessivelyelevated levels and sub-normal levels.

Transgenic mice were made that overexpressed mouse α2 alone, mouse β10alone or the mouse α2/β10 heterodimer (see example 6). Only thosetransgenics over expressing the α2/β10 heterodimer showed distinctphenotypic differences as compared to control mice. The α2/β10overexpressor transgenic mice exhibited a phenotype characterized bybilateral thyroid enlargement with multiple follicular papillaryadenomas and resulting hyperthyroidism, as indicated by elevated serumT4 levels. Other phenotypic changes were felt to be related to thesystemic hyperthyroid state, and included moderate hepatomegaly,hepatocellular hyperplasia, and slightly decreased serum cholesterollevels, bilateral renal hypertrophy, and a mild to moderate leukocytosiswith a predominance of lymphocytes (see example 6). Thus in a normalmouse setting α2/β10 clearly has a thyroid stimulating hormone (TSH)like activity. Due to the high level of amino acid conservation betweenmouse α2 and human α2 [88.5% identity and 90.4% similarity for thepredicted mature forms (i.e. without signal peptide)], the high level ofamino acid conservation between mouse β10 and human β10 [93.4% identityand 97.2% similarity for the predicted mature forms (i.e. without signalpeptide)], and the very high level of similarity between mouse thyroidgland biology and human thyroid gland biology, it is anticipated thathuman α2/β10 heterodimer has the same thyroid stimulating hormone (TSH)like activity as that found for the mouse α2/β10 heterodimer. Inaddition to TSH-like activity, α2/β10 may have other, distinct,biological effects in different physiological settings (i.e., diseaseconditions), as described in greater detail further herein.

TSH influences basal metabolism by regulating the production of thyroidhormones and is used clinically for enhancing the detection andtreatment of thyroid carcinoma; see McEvoy, G. (ed.), AHFS DrugInformation, pp. 2041-2042, American Society of Health-SystemPharmacists, Inc., Bethesda, Md. (1998). In addition, diagnostic testsfor measuring TSH levels in the blood are commonly used for determiningthe functional status of the thyroid gland when thyroid gland disorderis suspected. It is likely that human α2/β10 will have similar clinicalutilities as TSH and will be useful for the treatment and diagnosis ofthyroid gland related diseases and disorders. In addition, human α2/β10may have other therapeutic and diagnostic uses which are describedherein. It is reasonable to surmise that human α2/β10 selective bindingagents, for example, antibodies, will have similar clinical utilities toTSH selective binding agents and will therefore be useful for thetreatment and diagnosis of thyroid gland related diseases and disorders.In addition, human α2/β10 selective binding agents may have othertherapeutic and diagnostic uses as described herein.

Compositions and Administration

Therapeutic compositions are within the scope of the present invention.Such pharmaceutical compositions may comprise a therapeuticallyeffective amount of a β10 polypeptide, α2/β10 heterodimer or a β10nucleic acid molecule in admixture with a pharmaceutically orphysiologically acceptable formulation agent selected for suitabilitywith the mode of administration. Pharmaceutical compositions maycomprise a therapeutically effective amount of one or more β10polypeptide or α2/β10 heterodimer selective binding agents in admixturewith a pharmaceutically or physiologically acceptable formulation agentselected for suitability with the mode of administration.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed.

The pharmaceutical composition may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.Suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine),antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite), buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates, other organic acids), bulking agents(such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin or immunoglobulins),coloring, flavoring and diluting agents, emulsifying agents, hydrophilicpolymers (such as polyvinylpyrrolidone), low molecular weightpolypeptides, salt-forming counterions (such as sodium), preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such asglycerin, propylene glycol or polyethylene glycol), sugar alcohols (suchas mannitol or sorbitol), suspending agents, surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal), stability enhancing agents (sucrose orsorbitol), tonicity enhancing agents (such as alkali metal halides(preferably sodium or potassium chloride), mannitol sorbitol), deliveryvehicles, diluents, excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company [1990]).

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of β10 polypeptide or α2/β10 heterodimermolecule.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefor. In oneembodiment of the present invention, β10 polypeptide or α2/β10heterodimer compositions may be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents (Remington's Pharmaceutical Sciences, supra) in theform of a lyophilized cake or an aqueous solution. Further, the β10polypeptide or α2/β10 heterodimer product may be formulated as alyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of this invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.The preparation of such pharmaceutically acceptable compositions iswithin the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired β10 polypeptide or α2/β10 heterodimer molecule in apharmaceutically acceptable vehicle. A particularly suitable vehicle forparenteral injection is sterile distilled water in which a β10polypeptide or α2/β10 heterodimer molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), or beads, or liposomes, thatprovides for the controlled or sustained release of the product whichmay then be delivered as a depot injection. Hyaluronic acid may also beused, and this may have the effect of promoting sustained duration inthe circulation. Other suitable means for the introduction of thedesired molecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition may be formulated forinhalation. For example, a β10 polypeptide or α2/β10 heterodimermolecule may be formulated as a dry powder for inhalation. β10polypeptide, α2/β10 heterodimer or β10 nucleic acid molecule inhalationsolutions may also be formulated with a propellant for aerosol delivery.In yet another embodiment, solutions may be nebulized. Pulmonaryadministration is further described in PCT application no.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, β10 polypeptide orα2/β10 heterodimer molecules which are administered in this fashion canbe formulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Forexample, a capsule may be designed to release the active portion of theformulation at the point in the gastrointestinal tract whenbioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to facilitate absorption of the β10polypeptide or α2/β10 heterodimer molecule. Diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Another pharmaceutical composition may involve an effective quantity ofβ10 polypeptide or α2/β10 heterodimer molecules in a mixture withnon-toxic excipients which are suitable for the manufacture of tablets.By dissolving the tablets in sterile water, or other appropriatevehicle, solutions can be prepared in unit dose form. Suitableexcipients include, but are not limited to, inert diluents, such ascalcium carbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving β10 polypeptides or α2/β10heterodimers in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT/US93/00829 whichdescribes controlled release of porous polymeric microparticles for thedelivery of pharmaceutical compositions. Additional examples ofsustained-release preparations include semipermeable polymer matrices inthe form of shaped articles, e.g. films, or microcapsules. Sustainedrelease matrices may include polyesters, hydrogels, polylactides (U.S.Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). Sustained-release compositions also may include liposomes,which can be prepared by any of several methods known in the art. Seee.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985);EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using these methods may be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration may be stored in lyophilizedform or in solution. In addition, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the β10polypeptide or α2/β10 heterodimer molecule is being used, the route ofadministration, and the size (body weight, body surface or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician may titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage may range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage may range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the β10 polypeptide or α2/β10 heterodimer molecule in the formulationused. Typically, a clinician will administer the composition until adosage is reached that achieves the desired effect. The composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of the desired molecule)over time, or as a continuous infusion via implantation device orcatheter. Further refinement of the appropriate dosage is routinely madeby those of ordinary skill in the art and is within the ambit of tasksroutinely performed by them. Appropriate dosages may be ascertainedthrough use of appropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. oral, injection by intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes, or by sustained release systems orimplantation device. Where desired, the compositions may be administeredby bolus injection or continuously by infusion, or by implantationdevice.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or other appropriatematerial on to which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed release bolus, or continuousadministration.

In some cases, it may be desirable to use pharmaceutical compositionsaccording to this invention in an ex vivo manner. In such instances,cells, tissues, or organs that have been removed from the patient areexposed to pharmaceutical compositions after which the cells, tissuesand/or organs are subsequently implanted back into the patient.

In other cases, a β10 polypeptide or α2/β10 heterodimer of thisinvention can be delivered by implanting certain cells that have beengenetically engineered, using methods such as those described herein, toexpress and secrete the polypeptide or heterodimer. Such cells may beanimal or human cells, and may be autologous, heterologous, orxenogeneic. Optionally, the cells may be immortalized. In order todecrease the chance of an immunological response, the cells may beencapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

Additional embodiments of the present invention relate to cells andmethods (e.g., homologous recombination and/or other recombinantproduction methods) for both the in vitro production of therapeuticpolypeptides and for the production and delivery of therapeuticpolypeptides by gene therapy or cell therapy. Homologous and otherrecombination methods may be used to modify a cell that contains anormally transcriptionally silent β10 gene, or an under expressed gene,and thereby produce a cell which expresses therapeutically efficaciousamounts of β10 polypeptide or α2/β10 heterodimer.

Homologous recombination is a technique originally developed fortargeting genes to induce or correct mutations in transcriptionallyactive genes (Kucherlapati, Prog. in Nucl. Acid Res. & Mol. Biol.,36:301, 1989). The basic technique was developed as a method forintroducing specific mutations into specific regions of the mammaliangenome (Thomas et al., Cell, 44:419-428, 1986; Thomas and Capecchi,Cell, 51:503-512, 1987; Doetschman et al., Proc. Natl. Acad. Sci.,85:8583-8587, 1988) or to correct specific mutations within defectivegenes (Doetschman et al., Nature, 330:576-578, 1987). Exemplaryhomologous recombination techniques are described in U.S. Pat. No.5,272,071 (EP 9193051, EP Publication No. 505500; PCT/US90/07642,International Publication No. WO 91/09955).

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, therefore, recombine with otherpieces of endogenous DNA through shared homologous regions. If thiscomplementary strand is attached to an oligonucleotide that contains amutation or a different sequence or an additional nucleotide, it too isincorporated into the newly synthesized strand as a result of therecombination. As a result of the proofreading function, it is possiblefor the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA which mayinteract with or control the expression of a β10 polypeptide, e.g.,flanking sequences. For example, a promoter/enhancer element, asuppresser, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredβ10 polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of the desired β10polypeptide or α2/β10 heterodimer may be achieved not by transfection ofDNA that encodes the β10 gene itself, but rather by the use of targetingDNA (containing regions of homology with the endogenous gene ofinterest) coupled with DNA regulatory segments that provide theendogenous gene sequence with recognizable signals for transcription ofthe β10 gene.

In an exemplary method, the expression of a desired targeted gene in acell (i.e., a desired endogenous cellular gene) is altered viahomologous recombination into the cellular genome at a preselected site,by the introduction of DNA which includes at least a regulatorysequence, an exon and a splice donor site. These components areintroduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

Altered gene expression, as described herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, as well as increasing the expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained. The embodiments further encompass changing the pattern ofregulation or induction such that it is different from the pattern ofregulation or induction that occurs in the cell as obtained, andreducing (including eliminating) the expression of a gene which isexpressed in the cell as obtained.

One method by which homologous recombination can be used to increase, orcause, β10 polypeptide or α2/β10 heterodimer production from a cell'sendogenous β10 gene involves first using homologous recombination toplace a recombination sequence from a site-specific recombination system(e.g., Cre/loxP, FLP/FRT) (Sauer, Current Opinion In Biotechnology,5:521-527, 1994; Sauer, Methods In Enzymology, 225:890-900, 1993)upstream (that is, 5′ to) of the cell's endogenous genomic β10polypeptide coding region. A plasmid containing a recombination sitehomologous to the site that was placed just upstream of the genomic β10polypeptide coding region is introduced into the modified cell linealong with the appropriate recombinase enzyme. This recombinase causesthe plasmid to integrate, via the plasmid's recombination site, into therecombination site located just upstream of the genomic β10 polypeptidecoding region in the cell line (Baubonis and Sauer, Nucleic Acids Res.,21:2025-2029, 1993; O'Gorman et al., Science, 251:1351-1355, 1991). Anyflanking sequences known to increase transcription (e.g.,enhancer/promoter, intron, translational enhancer), if properlypositioned in this plasmid, would integrate in such a manner as tocreate a new or modified transcriptional unit resulting in de novo orincreased β10 polypeptide or α2/β10 heterodimer production from thecell's endogenous β10 gene.

A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic β10 polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, translocation) (Sauer, CurrentOpinion In Biotechnology, supra, 1994; Sauer, Methods In Enzymology,supra, 1993) that would create a new or modified transcriptional unitresulting in de novo or increased β10 polypeptide or α2/β10 heterodimerproduction from the cell's endogenous β10 gene.

An additional approach for increasing, or causing, the expression of theβ10 polypeptide from a cell's endogenous β10 gene involves increasing,or causing, the expression of a gene or genes (e.g., transcriptionfactors) and/or decreasing the expression of a gene or genes (e.g.,transcriptional repressors) in a manner which results in de novo orincreased β10 polypeptide production from the cell's endogenous β10gene. This method includes the introduction of a non-naturally occurringpolypeptide (e.g., a polypeptide comprising a site specific DNA bindingdomain fused to a transcriptional factor domain) into the cell such thatde novo or increased β10 polypeptide or α2/β10 heterodimer productionfrom the cell's endogenous β10 gene results.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) one or more targetingsequences; (b) a regulatory sequence; (c) an exon; and (d) an unpairedsplice-donor site. The targeting sequence in the DNA construct directsthe integration of elements (a)-(d) into a target gene in a cell suchthat the elements (b)-(d) are operatively linked to sequences of theendogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence of the β10 gene presented herein, a piece of DNA that iscomplementary to a selected region of the gene can be synthesized orotherwise obtained, such as by appropriate restriction of the native DNAat specific recognition sites bounding the region of interest. Thispiece serves as a targeting sequence(s) upon insertion into the cell andwill hybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be incorporated into the newly synthesized daughter strand ofDNA. The present invention, therefore, includes nucleotides encoding aβ10 polypeptide, which nucleotides may be used as targeting sequences.

β10 polypeptide or α2/β10 heterodimer cell therapy, e.g., theimplantation of cells producing β10 polypeptides or α2/β10 heterodimersis also contemplated. This embodiment involves implanting cells capableof synthesizing and secreting a biologically active form of the β10polypeptide or α2/β10 heterodimer. Such β10 polypeptide or α2/β10heterodimer-producing cells can be cells that are natural producers ofβ10 polypeptides or α2/β10 heterodimers or may be recombinant cellswhose ability to produce β10 polypeptides or α2/β10 heterodimers hasbeen augmented by transformation with a gene encoding the desired β10polypeptide or with a gene augmenting the expression of β10 polypeptideor α2/β10 heterodimer. Such a modification may be accomplished by meansof a vector suitable for delivering the gene as well as promoting itsexpression and secretion. In order to minimize a potential immunologicalreaction in patients being administered a β10 polypeptide or α2/β10heterodimer as may occur with the administration of a polypeptide of aforeign species, it is preferred that the natural cells producing β10polypeptide or α2/β10 heterodimer be of human origin and produce humanβ10 polypeptide or α2/β10 heterodimer. Likewise, it is preferred thatthe recombinant cells producing β10 polypeptide or α2/β10 heterodimer betransformed with an expression vector containing a gene encoding a humanβ10 polypeptide.

Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow the release of β10 polypeptide or α2/β10heterodimer but that prevent the destruction of the cells by thepatient's immune system or by other detrimental factors from thesurrounding tissue. Alternatively, the patient's own cells, transformedto produce β10 polypeptides or α2/β10 heterodimers ex vivo, may beimplanted directly into the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be routinely accomplished. For example, Baetge et al.(WO95/05452; PCT/US94/09299) describe membrane capsules containinggenetically engineered cells for the effective delivery of biologicallyactive molecules. The capsules are biocompatible and are easilyretrievable. The capsules encapsulate cells transfected with recombinantDNA molecules comprising DNA sequences coding for biologically activemolecules operatively linked to promoters that are not subject to downregulation in vivo upon implantation into a mammalian host. The devicesprovide for the delivery of the molecules from living cells to specificsites within a recipient. In addition, see U.S. Pat. Nos. 4,892,538,5,011,472, and 5,106,627. A system for encapsulating living cells isdescribed in PCT Application no. PCT/US91/00157 of Aebischer et al. Seealso, PCT Application no. PCT/US91/00155 of Aebischer et al., Winn etal., Exper. Neurol., 113:322-329 (1991), Aebischer et al., Exper.Neurol., 111:269-275 (1991); and Tresco et al., ASAIO, 38:17-23 (1992).

In vivo and in vitro gene therapy delivery of β10 polypeptides or α2/β10heterodimers is also envisioned. One example of a gene therapy techniqueis to use the β10 gene (either genomic DNA, cDNA, and/or synthetic DNA)encoding a β10 polypeptide which may be operably linked to aconstitutive or inducible promoter to form a “gene therapy DNAconstruct”. The promoter may be homologous or heterologous to theendogenous gene, provided that it is active in the cell or tissue typeinto which the construct will be inserted. Other components of the genetherapy DNA construct may optionally include, DNA molecules designed forsite-specific integration (e.g., endogenous sequences useful forhomologous recombination), tissue-specific promoter, enhancer(s) orsilencer(s), DNA molecules capable of providing a selective advantageover the parent cell, DNA molecules useful as labels to identifytransformed cells, negative selection systems, cell specific bindingagents (as, for example, for cell targeting), cell-specificinternalization factors, and transcription factors to enhance expressionby a vector as well as factors to enable vector manufacture.

A gene therapy DNA construct can then be introduced into cells (eitherex vivo or in vivo) using viral or non-viral vectors. One means forintroducing the gene therapy DNA construct is by means of viral vectorsas described herein. Certain vectors, such as retroviral vectors, willdeliver the DNA construct to the chromosomal DNA of the cells, and thegene can integrate into the chromosomal DNA. Other vectors will functionas episomes, and the gene therapy DNA construct will remain in thecytoplasm.

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the β10 gene in the target cell. Such elementsare turned on in response to an appropriate effector. In this way, atherapeutic polypeptide can be expressed when desired. One conventionalcontrol means involves the use of small molecule dimerizers or rapalogs(as described in WO9641865 (PCT/US96/099486); WO9731898 (PCT/US97/03137)and WO9731899 (PCT/US95/03157) used to dimerize chimeric proteins whichcontain a small molecule-binding domain and a domain capable ofinitiating biological process, such as a DNA-binding protein ortranscriptional activation protein. The dimerization of the proteins canbe used to initiate transcription of the transgene.

An alternative regulation technology uses a method of storing proteinsexpressed from the gene of interest inside the cell as an aggregate orcluster. The gene of interest is expressed as a fusion protein thatincludes a conditional aggregation domain which results in the retentionof the aggregated protein in the endoplasmic reticulum. The storedproteins are stable and inactive inside the cell. The proteins can bereleased, however, by administering a drug (e.g., small molecule ligand)that removes the conditional aggregation domain and thereby specificallybreaks apart the aggregates or clusters so that the proteins may besecreted from the cell. See, Science 287:816-817, and 826-830 (2000).

Other suitable control means or gene switches include, but are notlimited to, the following systems. Mifepristone (RU486) is used as aprogesterone antagonist. The binding of a modified progesterone receptorligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors which thenpass into the nucleus to bind DNA. The ligand binding domain is modifiedto eliminate the ability of the receptor to bind to the natural ligand.The modified steroid hormone receptor system is further described inU.S. Pat. No. 5,364,791; WO9640911, and WO9710337.

Yet another control system uses ecdysone (a fruit fly steroid hormone)which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain/DNA-bindingdomain/ligand-binding domain to initiate transcription. The ecdysonesystem is further described in U.S. Pat. No. 5,514,578; WO9738117;WO9637609; and WO9303162.

Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758; 5,650,298 and 5,654,168.

Additional expression control systems and nucleic acid constructs aredescribed in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

In vivo gene therapy may be accomplished by introducing the geneencoding a β10 polypeptide into cells via local injection of a β10nucleic acid molecule or by other appropriate viral or non-viraldelivery vectors. Hefti, Neurobiology, 25:1418-1435 (1994). For example,a nucleic acid molecule encoding a β10 polypeptide of this invention maybe contained in an adeno-associated virus (AAV) vector for delivery tothe targeted cells (e.g., Johnson, International Publication No.WO95/34670; International Application No. PCT/US95/07178). Therecombinant AAV genome typically contains AAV inverted terminal repeatsflanking a DNA sequence encoding a β10 polypeptide operably linked tofunctional promoter and polyadenylation sequences.

Alternative suitable viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631,236 involving adenoviralvectors; U.S. Pat. No. 5,672,510 involving retroviral vectors; and U.S.Pat. No. 5,635,399 involving retroviral vectors expressing cytokines.

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include the useof inducible promoters, tissue-specific enhancer-promoters, DNAsequences designed for site-specific integration, DNA sequences capableof providing a selective advantage over the parent cell, labels toidentify transformed cells, negative selection systems and expressioncontrol systems (safety measures), cell-specific binding agents (forcell targeting), cell-specific internalization factors, andtranscription factors to enhance expression by a vector as well asmethods of vector manufacture. Such additional methods and materials forthe practice of gene therapy techniques are described in U.S. Pat. No.4,970,154 involving electroporation techniques; WO96/40958 involvingnuclear ligands; U.S. Pat. No. 5,679,559 describing alipoprotein-containing system for gene delivery; U.S. Pat. No. 5,676,954involving liposome carriers; U.S. Pat. No. 5,593,875 concerning methodsfor calcium phosphate transfection; and U.S. Pat. No. 4,945,050 whereinbiologically active particles are propelled at cells at a speed wherebythe particles penetrate the surface of the cells and become incorporatedinto the interior of the cells.

It is also contemplated that β10 gene therapy or cell therapy canfurther include the delivery of one or more additional polypeptide(s) inthe same or a different cell(s). Such cells may be separately introducedinto the patient, or the cells may be contained in a single implantabledevice, such as the encapsulating membrane described above, or the cellsmay be separately modified by means of viral vectors.

A means to increase endogenous β10 polypeptide expression in a cell viagene therapy is to insert one or more enhancer elements into the β10polypeptide promoter, where the enhancer element(s) can serve toincrease transcriptional activity of the β10 gene. The enhancerelement(s) used will be selected based on the tissue in which onedesires to activate the gene(s); enhancer elements known to conferpromoter activation in that tissue will be selected. For example, if agene encoding a β10 polypeptide is to be “turned on” in T-cells, the lckpromoter enhancer element may be used. Here, the functional portion ofthe transcriptional element to be added may be inserted into a fragmentof DNA containing the β10 polypeptide promoter (and optionally, insertedinto a vector and/or 5′ and/or 3′ flanking sequence(s), etc.) usingstandard cloning techniques. This construct, known as a “homologousrecombination construct”, can then be introduced into the desired cellseither ex vivo or in vivo.

Gene therapy can also be used to decrease β10 polypeptide or α2/β10heterodimer expression by modifying the nucleotide sequence of theendogenous promoter(s). Such modification is typically accomplished viahomologous recombination methods. For example, a DNA molecule containingall or a portion of the promoter of the β10 gene(s) selected forinactivation can be engineered to remove and/or replace pieces of thepromoter that regulate transcription. For example the TATA box and/orthe binding site of a transcriptional activator of the promoter may bedeleted using standard molecular biology techniques; such deletion caninhibit promoter activity thereby repressing the transcription of thecorresponding β10 gene. The deletion of the TATA box or thetranscription activator binding site in the promoter may be accomplishedby generating a DNA construct comprising all or the relevant portion ofthe β10 polypeptide promoter(s) (from the same or a related species asthe β10 gene(s) to be regulated) in which one or more of the TATA boxand/or transcriptional activator binding site nucleotides are mutatedvia substitution, deletion and/or insertion of one or more nucleotides.As a result, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. The construct willtypically contain at least about 500 bases of DNA that correspond to thenative (endogenous) 5′ and 3′ DNA sequences adjacent to the promotersegment that has been modified. The construct may be introduced into theappropriate cells (either ex vivo or in vivo) either directly or via aviral vector as described herein. Typically, the integration of theconstruct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

Other Uses of the Nucleic Acids and Polypeptides of this Invention

Nucleic acid molecules of the present invention (including those that donot themselves encode biologically active polypeptides) may be used tomap the locations of the β10 gene and related genes on chromosomes.Mapping may be done by techniques known in the art, such as PCRamplification and in situ hybridization.

β10 nucleic acid molecules (including those that do not themselvesencode biologically active polypeptides), may be useful as hybridizationprobes in diagnostic assays to test, either qualitatively orquantitatively, for the presence of a β10 DNA or corresponding RNA inmammalian tissue or bodily fluid samples.

The β10 polypeptides or α2/β10 heterodimers may be used (simultaneouslyor sequentially) in combination with one or more cytokines, growthfactors, antibiotics, anti-inflammatories, and/or chemotherapeuticagents as is appropriate for the indication being treated.

Other methods may also be employed where it is desirable to inhibit theactivity of one or more β10 polypeptides or α2/β10 heterodimers of thisinvention. Such inhibition may be effected by nucleic acid moleculeswhich are complementary to and hybridize to expression control sequences(triple helix formation) or to β10 mRNA. For example, antisense DNA orRNA molecules, which have a sequence that is complementary to at least aportion of the selected β10 gene(s) can be introduced into the cell.Anti-sense probes may be designed by available techniques using thesequence of the β10 polypeptide disclosed herein. Typically, each suchantisense molecule will be complementary to the start site (5′ end) ofeach selected β10 gene. When the antisense molecule then hybridizes tothe corresponding β10 mRNA, translation of this mRNA is prevented orreduced. Anti-sense inhibitors provide information relating to thedecrease or absence of a β10 polypeptide or α2/β10 heterodimer in a cellor organism.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more β10 polypeptides or α2/β10heterodimers. In this situation, the DNA encoding a mutant polypeptideof each selected β10 polypeptide can be prepared and introduced into thecells of a patient using either viral or non-viral methods as describedherein. Each such mutant is typically designed to compete withendogenous polypeptide or heterodimer in its biological role.

In addition, a β10 polypeptide or α2/β10 heterodimer of this invention,whether biologically active or not, may be used as an immunogen, thatis, the polypeptide contains at least one epitope to which antibodiesmay be raised. Selective binding agents that bind to a β10 polypeptideor α2/β10 heterodimer (as described herein) may be used for in vivo andin vitro diagnostic purposes, including, but not limited to, use inlabeled form to detect the presence of β10 polypeptide or α2/β10heterodimer in a body fluid or cell sample. The antibodies may also beused to prevent, treat, or diagnose a number of diseases and disorders,including those recited herein. The antibodies may bind to a β10polypeptide or α2/β10 heterodimer so as to diminish or block at leastone activity characteristic of a β10 polypeptide or α2/β10 heterodimer,or may bind to a polypeptide to increase at least one activitycharacteristic of a β10 polypeptide or α2/β10 heterodimer (including byincreasing the pharmacokinetics of a β10 polypeptide or α2/β10heterodimer).

cDNA encoding human β10 polypeptide in E. coli was deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on Dec. 28, 1999, under accession numberPTA-1210.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

Example 1 DNA Encoding Human Beta-10

The amino acid sequence of CG-(chorionic gonadotropin)-β-subunit wasBlasted against an, in house generated, Virtual Protein database derivedfrom public human genomic sequences present in GenBank. A virtualprotein containing a 45 amino acid region with significant homology tothe carboxy half of CG-β was identified. The short (135 base pair)region of human genomic sequence that encoded the 45 amino acid stretchcame from an over 160 kilobase pair GenBank human genomic DNA sequence(accession # AL049871). By analyzing an 8 kilobase pair stretch ofgenomic sequence just 5′ of the 135 base pair sequence, a region havingsignificant homology (and containing a frameshift) to the N-terminalhalf of CG-β was identified. The nucleotide sequence of this novel genewas compiled from the genomic sequences. The amino acid sequence of thiscompiled gene had significant homology to the four known humanglycoprotein hormone β-subunit polypeptides and had an N-terminalpredicted signal peptide, consistent with this novel human gene being anew β-like member of the glycoprotein hormone family. There was a 4.5-kbintron located between the two putative N-terminal half and C-terminalhalf coding exons. Intron spanning PCR (see Tissue Expression, Beta-10section below) of cDNAs from various tissues sources revealed that β10was expressed in numerous tissues including pituitary.

The full coding region (ATG to TGA stop codon) and some of the 3′UTR(untranslated region) of β10 was cloned as one fragment by PCR using thefollowing reaction mix and PCR conditions:

Template: ten microliters of Human Pituitary Marathon Ready cDNA(Clontech Laboratories, Inc., Palo Alto, Calif.; catalog no. 7424-1).

(SEQ ID NO: 4) Forward primer: 5′-ATGAAGCTGGCATTCCTCTTCCTT-3′. (SEQ IDNO: 5) Reverse primer: 5′-GCATGTGCTGCTCACACAGGT-3′.Final concentration of each primer: 1.0 micromolar.Final concentration of dNTPs: 200 micromolar.Five units of Pfu polymerase (Stratagene, La Jolla, Calif.).Ten microliters of 10× Pfu reaction buffer (Stratagene, La Jolla,Calif.).Ten microliters of GC melt (Clontech Laboratories, Inc., Palo Alto,Calif.; Advantage GC cDNA PCR kit; catalog no. K1907-1).Final reaction volume: 100 microliters.Cycling conditions: 94° C. for sixty seconds followed by 45 cycles of94° C. (ten seconds), 60° C. (twenty seconds), 72° C. (ninety seconds),and then at the end of the 45th cycle an incubation at 72° C. for sevenminutes, then an additional 10 cycles of 94° C. (ten seconds), 60° C.(twenty seconds), 72° C. (ninety seconds), and then at the end of theadditional 10th cycle an incubation at 72° C. for seven minutes.

The PCR reaction was run on an agarose gel, and a single band was seen.This DNA band was cloned into pPCR-Script AMP (Stratagene). The sequenceof the insert in one of the resulting clones is that of SEQ ID NO: 2which contains the full coding region (ATG to TGA stop codon) and someof the 3′UTR (untranslated region) of β10.

The following is a list and description of β10 sequences from publiclyavailable databases:

GenBank Accession # AL049871: 170 kilobase pairs of human genomicsequence. No exons, genes or homologies are identified in this recordand the full coding region sequence of β10 is broken up by an intronicsequence.

GenBank Accession # AL118555: 126 kilobase pairs of human genomicsequence. No exons, genes or homologies are identified in this recordand the full coding region sequence of β10 is broken up by an intronicsequence.

Example 2 Tissue Expression, Beta-10

Using a PCR fragment as a probe, it was not possible to obtain ahybridization signal on various Human Multiple Tissue Northern Blots(Clontech Inc., Palo Alto, Calif.). Intron spanning PCR was used todetermine the expression pattern of beta-10 as described below.

For the Human Sure-RACE Panels (OriGene Technologies, Inc., Rockville,Md.; catalog no. HRAA-101) the cDNA samples represented brain, heart,kidney, spleen, liver, colon, lung, small intestine, muscle, stomach,testis, placenta, pituitary, thyroid gland, adrenal gland, pancreas,ovary, uterus, prostate, peripheral blood leucocytes, fetal brain, fetalliver, fat and mammary gland. Each cDNA sample was in a separate tube inthe form of a dried down pellet of DNA. The reaction mixture wascomposed as follows:

(SEQ ID NO: 6) Forward primer: 5′-CTGCAGGTGCCTTCGGATC-3′; (SEQ ID NO: 5)Reverse primer: 5′-GCATGTGCTGCTCACACAGGT-3′;Amount of each primer: 0.5 picomoles;Final concentration of dNTPs: 200 micromolar;2.5 microliters of GC melt (Clontech Laboratories, Inc., Palo Alto,Calif.; Advantage GC cDNA PCR kit; catalog no. K1907-1);2.5 units of Taq (Boehringer Mannheim, Indianapolis, Ind.; PCR Core Kit;catalog no. 1578 553);2.5 microliters of 10×PCR-reaction buffer (Boehringer Mannheim,Indianapolis, Ind.; PCR Core Kit; catalog no. 1578 553);

For each cDNA sample the above reaction mixture was made up to a volumeof 25 microliters, and 20 microliters of this mixture was added to thedried down cDNA pellet. The PCR conditions were as follows: 94° C. forsixty seconds, followed by 5 cycles of 94° C. (ten seconds) and 72° C.(forty seconds), followed by 5 cycles of 94° C. (ten seconds) and 70° C.(forty seconds), followed by 35 cycles of 94° C. (ten seconds) and 68°C. (forty seconds), and then followed by 68° C. for seven minutes.

PCR products were then analyzed by agarose gel electrophoresis. Thecorrect size PCR product of 293 base pairs, indicating expression ofβ10, was found in colon, small intestine, testis, pituitary and fetalliver.

For the Human Rapid-Scan Plate (OriGene Technologies, Inc., RockvilleMd.; catalog no. HSCA-101) the cDNA samples represented brain, heart,kidney, spleen, liver, colon, lung, small intestine, muscle, stomach,testis, placenta, salivary gland, thyroid gland, adrenal gland,pancreas, ovary, uterus, prostate, skin, peripheral blood leucocytes,bone marrow, fetal brain and fetal liver. Each cDNA sample was in aseparate tube in the form of a dried down pellet of DNA.

The reaction mixture that was utilized was as follows:

(SEQ ID NO: 6) Forward primer: 5′-CTGCAGGTGCCTTCGGATC-3′; (SEQ ID NO: 5)Reverse primer: 5′-GCATGTGCTGCTCACACAGGT-3′;Amount of each primer: 0.5 picomoles;Final concentration of dNTPs: 200 micromolar;2.5 microliters of GC melt (Clontech Laboratories, Inc., Palo Alto,Calif.; Advantage GC cDNA PCR kit; catalog no. K1907-1);2.5 units of Taq (Boehringer Mannheim, Indianapolis, Ind.; PCR Core Kit;catalog no. 1578 553);2.5 microliters of 10×PCR-reaction buffer (Boehringer Mannheim,Indianapolis, Ind.; PCR Core Kit; catalog no. 1578 553);

For each cDNA sample the above reaction mixture was made up to a volumeof 25 microliters, and then 20 microliters of this mixture was added tothe dried down cDNA pellet. The PCR conditions were as follows: 94° C.for sixty seconds followed by 5 cycles of 94° C. (ten seconds), 72° C.(forty seconds) and then followed by 5 cycles of 94° C. (ten seconds),70° C. (forty seconds) and then followed by 35 cycles of 94° C. (tenseconds), 68° C. (forty seconds) and then followed by 68° C. for sevenminutes.

PCR products were then analyzed by agarose gel electrophoresis. Thecorrect size PCR product of 293 base pairs, indicating expression ofβ10, was found in brain, spleen, liver, colon, stomach, placenta,thyroid gland, adrenal gland, pancreas, skin and peripheral bloodleucocytes.

Combining the expression results from the Human Sure-RACE Panels and theHuman Rapid-Scan Plate indicated that β10 is expressed in brain, liver,fetal liver, stomach, pituitary, colon, small intestine, thyroid gland,adrenal gland, pancreas, skin, peripheral blood leucocytes, spleen,testis and placenta.

Example 3 Tissue Expression, α2

Northern analysis was carried out to determine the expression pattern ofalpha-2. The probe for the Northerns was a 390-base pair PCR product(corresponding to nucleotides 56-445 of SEQ ID NO: 1 from WO99/41377).This PCR product was generated via a 466-base pair PCR intermediate asfollows:

PCR was first used to clone a 466-base pair fragment of alpha-2 fromhuman testis cDNA using the following reaction mixture and PCRconditions:

Template: ten microliters of Human Testis Marathon Ready cDNA (ClontechLaboratories, Inc., Palo Alto, Calif.; catalog no. 7414-1);

(SEQ ID NO: 7) Forward primer: 5′-GAGACATCTCCCCACTGTGTTT-3′; (SEQ ID NO:8) Reverse primer: 5′-GTTTCCCCCAACAGAATGTCAA-3′;Final concentration of each primer: 1.0 micromolar;Final concentration of dNTPs: 200 micromolar;Five units of Pfu polymerase;Final reaction volume: 100 microliters;Cycling conditions: 94° C. for sixty seconds followed by 35 cycles of94° C. (ten seconds), 60° C. (thirty seconds), 72° C. (sixty seconds),and then at the end of the 35th cycle an incubation at 72° C. for fiveminutes.

The PCR reaction was run on an agarose gel, and four distinct bands wereseen. The multiple bands arose from PCR amplification of contaminatinghuman genomic DNA present in the Human Testis Marathon Ready cDNA. The466-base pair PCR product was isolated from the agarose gel and cloned.A plasmid clone containing the 466-base pair sequence was used as atemplate for generating the 390-base pair PCR fragment using thefollowing reaction mix and PCR conditions:

Template: ten picograms of the plasmid clone containing the abovementioned 466-base pair sequence;

Forward primer: (SEQ ID NO: 9) 5′-ATGCCTATGGCGTCCCCTCAAAC-3′; Reverseprimer: (SEQ ID NO: 10) 5′-CTAGTAGCGAGAGAGGCGACACATGTCA-3′;Final concentration of each primer: 1.0 micromolar.Final concentration of dNTPs: 200 micromolar;Ten units of Taq polymerase;Final reaction volume: 100 microliters;Cycling conditions: 94° C. for sixty seconds, followed by 35 cycles of94° C. (ten seconds), 68° C. (sixty seconds), and then at the end of the35th cycle an incubation at 68° C. for six minutes.

The 390-base pair PCR product was then purified by agarose gelelectrophoresis. This PCR fragment was labeled with ³²P and hybridizedto various Clontech Human Multiple Tissue Northern Blots (tissues/cellsrepresented were: pancreas, adrenal medulla, thyroid, adrenal cortex,testis, thymus, small intestine, stomach, spleen, prostate, ovary,colon, peripheral blood leucocytes, brain, heart, skeletal muscle,kidney, liver, placenta and lung) and to a Northern blot made withpituitary mRNA using high stringency conditions as follows:

Hybridization was for one hour at 68° C. using Clontech “ExpressHybHybridization Solution”. The blots were washed in 2×SSC, 0.1% SDS atroom temperature twice, for twenty minutes each time. The blots werethen washed in 0.1×SSC, 0.1% SDS at 50° C. for ten minutes, and thenexposed to film.

A strong signal representing a single band was obtained in the pancreasmRNA lane and the pituitary mRNA lane. A significantly weaker signal wasseen in the placenta mRNA lane.

In situ hybridization was done to further determine sites of α2 geneexpression. A panel of normal embryonic (E10.5 through E18.5) and adultmouse tissues and adult rhesus monkey tissues were fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned at 5 micrometers.Prior to in situ hybridization, tissues were permeabilized with 0.2MHCL, followed by digestion with Proteinase K and acetylation withtriethanolamine and acetic anhydride. Sections were hybridized overnightat 55° C. with a ³³P-labeled antisense RNA probe complementary to eitherthe mouse or human (for rhesus tissues) α2 sequence and with sense(control) probes. The antisense and sense ³³P-labeled RNA probes wereobtained by in vitro transcription of plasmid DNAs containing either themouse α2 cDNA (bacterial clone no. 1224990 from the public WashU-HHMIMouse EST Project) or the human α2 cDNA (plasmid clone containing theabove described PCR generated 390-base pair human α2 coding regionsequence).

Following hybridization, sections were washed in buffer, treated withRNaseA to remove unhybridized probe, and then subjected to a highstringency wash in 0.1×SSC at 55° C. Slides were dipped in Kodak NTB2emulsion, exposed at 4° C. for two-three weeks, developed, and thencounterstained. Sections were examined with darkfield and standardillumination to allow simultaneous evaluation of tissue morphology andhybridization signal. The following tissues were then examined:

Mouse tissues: Brain (1 sagittal, 2 coronal sections); GI tract(esophagus, stomach, duodenum, jejunum, ileum, proximal & distal colon);pituitary; liver; lung; heart; spleen; thymus; lymph nodes; kidney;adrenal; bladder; pancreas; salivary gland; male and female reproductiveorgans (ovary, oviduct and uterus in the female; testis, epididymus,prostate, seminal vesicle and vas deferens in the male); BAT & WAT(subcutaneous, peri-renal); bone (femur); skin; breast; and skeletalmuscle.

Rhesus tissues: adrenal gland; liver; gall bladder; intestine; pancreas;and salivary gland.

Both mouse and human antisense probes produced positive signaldetectable above a very low level of background seen with the sensestrand controls. In the embryonic mouse, no signal was observed in anymajor organs from E8.5 through E18.5. At E15.5 and E18.5, signal waspresent over scattered cells adjacent to some of the developing bones ofthe head and teeth. In the adult mouse, a moderate level of signal waspresent in the adrenal cortex. A lower level of signal was detectable inthe anterior and intermediate lobes of the pituitary as well as inintestinal epithelium at the level of the crypts. In addition, graindensity was slightly above background in developing sperm within theseminiferous tubules of the testis and in granulosa cells surroundingdeveloping follicles in the ovary.

In rhesus tissues, moderate signal was noted in the adrenal cortex, gallbladder epithelium, and in the intestinal epithelium primarily at thelevel of the crypts.

Combining the expression results from the α2 Northerns and the α2 insitu analysis indicates that α2 is expressed in anterior pituitary,placenta, pancreas, adrenal cortex, intestinal crypts and gall bladdermucosa.

Example 4 Antibodies Against α2

Rabbit polyclonal antibodies were generated against α2 by immunizingrabbits with peptide CSPRYSVLVASGYRHN (SEQ ID NO: 28) that had beenconjugated to Keyhole Limpet Hemocyanin (cat#77605 Pierce Inc.,Rockford, Ill.). The peptide was synthesized with a C-terminal amide sorather than the C-terminus being COOH the C-terminus was CONH₂. TheRYSVLVASGYRHN portion of the peptide sequence is totally conservedbetween human and mouse α2. This region was chosen so that theantibodies would be able to bind to human α2 and mouse α2. Theantibodies were affinity purified from rabbit serum over a column(SulfoLink Kit, cat#44895, Pierce Inc., Rockford, Ill.) to which thepeptide antigen (SEQ ID NO: 28) had been attached. Western blot analysisof conditioned media harvested from 293 cells that had been transfectedwith either a human α2-polyHis-tag mammalian expression vector or ahuman alpha-subunit-polyHis-tag mammalian expression vector demonstratedthat these affinity purified polyclonal antibodies had high specificityfor α2 polypeptide and did not cross react with alpha-subunit.

Example 5 DNA Encoding Mouse Beta-10

Various human Beta-10 cDNA probes were used to probe a mouse genomic129SvJ BAC library arrayed on high density filters (catalog#FBAC-4431,Genome Systems, St. Louis, Mo.). The mouse BAC clone inplate#218-well#P22 was obtained (catalog# FBAC-4432, Genome Systems, St.Louis, Mo.). A 10-kb HindIII sub-fragment from this BAC clone hybridizedstrongly to a human Beta-10 cDNA probe. This 10-kb HindIII fragment wassubcloned into pBluescriptII-KS(−) and fully sequenced. Computationalanalysis of this 10-kb mouse genomic sequence was used to identify twoexons encoding the mouse ortholog of human Beta-10.

Primers were designed from this electronic sequence to clone the mouseBeta-10 cDNA as follows:

Template: twenty microliters of mouse testis Marathon Ready cDNA(Clontech Laboratories, Inc., Palo Alto, Calif.; catalog no. 7455-1).

Forward primer: (SEQ ID NO: 14)5′-ATTACTAGTTCCACCATGAAGTTGGTATACCTTGTCCTT-3′; Reverse primer: (SEQ IDNO: 15) 5′-TTAATAATCGATCGTCAGATGGTCTCACACTCAGTG-3′;Final concentration of each primer: 1.0 micromolar.PCR kit: Expand High Fidelity PCR System (catalog#1732641, BoehringerMannheim Corporation, Indianapolis, Ind.).Final reaction volume: 50 microliters;Cycling conditions: 94° C. for sixty seconds, followed by 55 cycles of94° C. (ten seconds), 65° C. (twenty seconds), 72° C. (forty seconds),and then at the end of the 55th cycle an incubation at 72° C. for sevenminutes.

The 0.4-kb PCR product was then purified by agarose gel electrophoresisand cloned into pCR2.1 (Invitrogen Inc., Carlsbad, Calif.). A clonecontaining the full coding region cDNA of mouse Beta-10 (SEQ ID NO: 12)was identified by sequencing.

Example 6 Production and Analysis of Transgenic Mice Over Expressing α2Alone, β10 Alone and Co-Expressing α2 and β10

Transgenic mice over expressing α2 alone, β10 alone and co-expressing α2and β10 from the human apolipoprotein E promoter were generatedessentially as previously described (Simonet et al, 1997, Cell vol 89 p309-319). This human apoE promoter vector directs high level, liverspecific, gene expression in transgenic mice, and has been previouslyused to generate transgenic mice having high levels of transgene encodedsecreted protein in their circulation. For all phenotypic analysesdescribed below the non-transgenic controls were mice that were producedduring the same series of microinjections as the transgenic expressorsin question.

Genomic (i.e. containing α2 and β10 exons and introns) transgenes wereused for generating transgenics to maximize expression. Additionally, inall cases a Kozak site (CCACC) was engineered just 5′ of the start siteATG.

The human apoE promoter expression vector contains the HCR (liverspecific enhancer element) followed by the apoE promoter and firstintron (in apoE 5′UTR) and then by the SV40 polyA-signal/terminator.There are unique SpeI and SfiI (compatible with PvuI) sites fordirectional cloning of genes in between the apoE promoter-first intronand the SV40 polyA-signal/terminator regions of the vector.

The following procedure was used to generate the “mouse genomic α2 humanapoE expression vector transgene”:

Pools of the BAC Mouse ES (129SvJ) Genomic Screening Kit(catalog#BDTW-7460, Genome Systems, St. Louis, Mo.) were screened by PCRusing mouse α2 specific primers. The mouse BAC clone inplate#44-well#H19 was obtained (catalog# FBAC-4432, Genome Systems, St.Louis, Mo.). A 12-kb XbaI sub-fragment from this BAC clone hybridizedstrongly to a mouse α2 cDNA probe. This 12-kb XbaI fragment wassubcloned into pBluescriptII-KS(−)(Stratagene Inc, La Jolla, Calif.) andfully sequenced. Computational analysis of this 12-kb mouse genomicsequence was used to identify the three α2 coding exons.

Primers were designed to amplify the complete mouse genomic α2 codingsequence (start ATG to stop TAG; SEQ ID NO: 18) as follows:

Template: 5 nanograms of the 12-kb XbaI pBluescriptII-KS(−) clone DNA.

Forward primer: (SEQ ID NO: 16)5′-CCGCACTAGTTCCACCATGCCCATGGCACCACGAGT-3′; Reverse primer: (SEQ ID NO:17) 5′-GCGGCGTTCGATCGCTAGTAGCGGGAGAAACGGCACATATC-3′;Final concentration of each primer: 1.0 micromolar.PCR kit: Pfu DNA Polymerase kit (Stratagene, La Jolla, Calif.).Final reaction volume: 50 microliters;Cycling conditions: 94° C. for sixty seconds, followed by 40 cycles of94° C. (ten seconds), 60° C. (twenty seconds), 72° C. (180 seconds), andthen at the end of the 40th cycle an incubation at 72° C. for fourminutes.

The 0.8-kb PCR product was purified by agarose gel electrophoresis, cutwith SpeI and PvuI and cloned into SpeI-SfiI cut human apoE expressionvector. A clone containing the accurate full genomic coding region mouseα2 was identified by sequencing. DNA from this “mouse genomic α2 humanapoE expression vector transgene” clone was digested with ClaI andApaLI; the 4-kb band was purified by gel electrophoresis and used togenerated transgenic mice as previously described (Simonet et al, 1997,Cell vol 89 p 309-319).

Transgenic mice were identified by PCR as follows: Template: ear punchDNA.

Forward primer: 5′-GCCTCTAGAAAGAGCTGGGAC-3′; (SEQ ID NO: 19) Reverseprimer: 5′-CGCCGTGTTCCATTTATGAGC-3′; (SEQ ID NO: 20)Final concentration of each primer: 1.0 micromolar.PCR kit: Ready-to-Go PCR Beads (Amersham Pharmacia Biotech Inc.,Piscataway, N.J.).Final reaction volume: 25 microliters;Cycling conditions: 30 cycles of 94° C. (sixty seconds), 62° C. (twentyseconds), 72° C. (30 seconds).

Upon electrophoresis of the PCR product the presence of the 0.37-kb bandindicated that the particular mouse was transgenic.

Transgenics over expressing α2 were identified by Western blot of plasma(using the affinity purified anti-α2 polyclonal antibody described abovein Example 4) obtained from the various PCR identified transgenics. Sixα2 overexpressors (#s 7, 8, 26, 28, 156 and 186) as well as sixnon-transgenic control mice (#s 10, 11, 12, 18, 20, 21) werephenotypically analyzed. All mice were injected with 50 mg/kg BrdU onehour prior to harvest, radiographed, and sacrificed. Mice weresacrificed at 12 weeks of age. No significant findings were noted duringnecropsy.

For all mice, body and selected organ weights were taken, blood wasdrawn for hematology and serum chemistries, and organs were harvestedfor histologic analysis and BrdU labeling.

H&E stained sections of liver, gall bladder, spleen, lung, brain,pituitary, heart, kidney, adrenal, stomach, small intestine, pancreas,cecum, colon, mesenteric lymph node, skin, mammary gland, trachea,esophagus, thyroid, parathyroid, salivary gland, urinary bladder, ovaryor testis, uterus or prostate and seminal vesicle, bone, and bone marrowwere examined.

There were no biologically meaningful differences in the mean orindividual animal organ weights, hematology values, clinical chemistryvalues or histologic findings between the α2 overexpressor transgenicmice and the non-transgenic control mice. In other words the α2overexpressor transgenic mice did not have a phenotype.

The following procedure was used to generate the “mouse genomic β10human apoE expression vector transgene”.

Primers were designed to amplify the complete mouse genomic β10 codingsequence (start ATG to stop TGA; SEQ ID NO: 23) as follows:

Template: 10 nanograms of the mouse genomic β10 10-kb HindIIIpBluescriptII-KS(−) clone DNA described in example 5.

Forward primer: (SEQ ID NO: 21)5′-ATTACTAGTTCCACCATGAAGTTGGTATACCTTGTCCTT-3′; Reverse primer: (SEQ IDNO: 22) 5′-TTAATAATCGATCGTCAGATGGTCTCACACTCAGTG-3′;Final concentration of each primer: 1.0 micromolar.PCR kit: PfuTurbo DNA Polymerase kit (Stratagene, La Jolla, Calif.).Final reaction volume: 50 microliters;Cycling conditions: 92° C. for sixty seconds, followed by 15 cycles of92° C. (ten seconds), 65° C. (twenty seconds), 68° C. (four minutes).

The 3-kb PCR product was purified by agarose gel electrophoresis, cutwith SpeI and PvuI and cloned into SpeI-SfiI cut human apoE expressionvector. A “mouse genomic β10 human apoE expression vector transgene”clone containing the accurate full genomic coding region mouse β10 wasidentified by sequencing.

The following procedure was then used to generate the combined “mousegenomic β10 mouse genomic α2 human apoE expression vector transgene”.

“Mouse genomic β10 human apoE expression vector transgene” clone DNA wascut with HindIII and SacII, and the ends were made blunt with DNApolymerase I Large (Klenow) Fragment (New England Biolabs, Beverly,Mass.). The 5.6-kb fragment was gel purified and cloned into HincII cutpBluescript II KS(−). A clone with the 5.6-kb “mouse genomic β10 humanapoE expression cassette” in the orientation that has the SV40polyA-signal/terminator region of the cassette next to HindIII site inthe pBluescript II KS(−) polylinker was identified. DNA from this clonewas cut with HindIII and SacII, and ligated to the 3.4-kb HindIII-SacII“mouse genomic α2 human apoE expression cassette” that had been isolatedfrom the “mouse genomic α2 human apoE expression vector transgene” clonedescribed above. The final 11.8-kb “mouse genomic β10 mouse genomic α2human apoE expression vector transgene” construct consists of the “mousegenomic β10 human apoE expression cassette” and the “mouse genomic α2human apoE expression cassette” cloned in tandem (i.e. both in the sametranscriptional orientation) into pBluescript II KS(−). In thisconstruct β10 and α2 each have their own HCR/apoE promoter and SV40polyA-signal/terminator for expression purposes.

DNA from this “mouse genomic β10 mouse genomic α2 human apoE expressionvector transgene” clone was digested with BssHII; the 9-kb band waspurified by gel electrophoresis and used to generated transgenic mice aspreviously described (Simonet et al, 1997, Cell vol 89 p 309-319).

Mice transgenic for the “mouse genomic α2 human apoE expressioncassette” were identified by PCR as follows:

Template: ear punch DNA.

Forward primer: 5′-CCAGTGTGATATGTGCCGTTTC-3′; (SEQ ID NO: 24) Reverseprimer: 5′-GAAGAGCGCAGAGCTCGGTA-3′; (SEQ ID NO: 25)Final concentration of each primer: 1.0 micromolar.PCR kit: Ready-to-Go PCR Beads (Amersham Pharmacia Biotech Inc.,Piscataway, N.J.).Final reaction volume: 25 microliters;Cycling conditions: 94° C. for sixty seconds, followed by 35 cycles of94° C. (ten seconds), 60° C. (twenty seconds), 72° C. (forty seconds),and then at the end of the 35th cycle an incubation at 72° C. for sevenminutes.

The forward primer [5′-CCAGTGTGATATGTGCCGTTTC-3′ (SEQ ID NO: 24)] forthis PCR is located in the 3^(rd) α2 coding exon (this exon contains thestop codon).

The reverse primer [5′-GAAGAGCGCAGAGCTCGGTA-3′ (SEQ ID NO: 25)] for thisPCR is located in the SV40 polyA-signal/terminator region.

Upon electrophoresis of the PCR product the presence of the 0.31-kb bandindicated that the particular mouse was transgenic for the “mousegenomic α2 human apoE expression cassette”. Those mouse numbers were:25, 45, 53, 76, 94, 95, and 113.

Mice transgenic for the “mouse genomic β10 human apoE expressioncassette” were identified by PCR as follows:

Template: ear punch DNA.

Forward primer: 5′-TGGAGTCGATCCTTTCTACACCTA-3′; (SEQ ID NO: 26) Reverseprimer: 5′-AGAGCGCAGAGCTCGGTAC-3′; (SEQ ID NO: 27)Final concentration of each primer: 1.0 micromolar.PCR kit: Ready-to-Go PCR Beads (Amersham Pharmacia Biotech Inc.,Piscataway, N.J.).Final reaction volume: 25 microliters;Cycling conditions: 94° C. for sixty seconds, followed by 35 cycles of94° C. (ten seconds), 60° C. (twenty seconds), 72° C. (forty seconds),and then at the end of the 35th cycle an incubation at 72° C. for sevenminutes.

The forward primer [5′-TGGAGTCGATCCTTTCTACACCTA-3′ (SEQ ID NO: 26)] forthis PCR is located in the 2nd β10 coding exon (this exon contains thestop codon).

The reverse primer [5′-AGAGCGCAGAGCTCGGTAC-3′ (SEQ ID NO: 27)] for thisPCR is located in the SV40 polyA-signal/terminator region.

Upon electrophoresis of the PCR product the presence of the 0.37-kb bandindicated that the particular mouse was transgenic for the “mousegenomic β10 human apoE expression cassette”. Those mouse numbers were:25, 31, 45, 53, 76, 94, 95, and 113. Of note, mouse #31 which waspositive by PCR for the “mouse genomic β10 human apoE expressioncassette” was negative by PCR for the “mouse genomic α2 human apoEexpression cassette”.

Mouse #76 and #113 died shortly after the PCR genotyping. The remainingsix transgenics [#s 25 (female), 31 (male), 45 (female), 53 (male), 94(male), and 95 (male)] as well as five non-transgenic control mice [#s17 (male), 18 (female), 19 (female), 20 (male) and 21 (male)] werenecropsied at 7 weeks of age for subsequent phenotypical analysis. Allmice were injected with 50 mg/kg BrdU one hour prior to harvest,radiographed, and sacrificed. Upon necropsy abnormally large thyroidglands were found in some of the transgenic mice. As part of thenecropsy, mice were weighed, blood was drawn for hematology and serumchemistries, and liver, spleen, kidney, heart, and thymus were weighed.Sections of liver, gall bladder, spleen, lung, brain, pituitary, heart,kidney, adrenal, thymus, stomach, small intestine, pancreas, cecum,colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus,thyroid, parathyroid, salivary gland, urinary bladder, ovary or testis,uterus or prostate and seminal vesicle, bone, and bone marrow wereharvested for histologic analysis.

Northern blot analysis was used to determine the levels of α2 and β10mRNA in the livers of all of the transgenic and non-transgenic controlmice as described below.

Total RNA was isolated from liver samples, quantitated and 10 microgramsof total RNA for each mouse was electrophoresed in a formaldehydedenaturation agarose gel and transferred to a Nylon membrane. This wasdone in duplicate to generate 2 Northern blots for probing. EthidiumBromide staining of the agarose gels revealed virtually equal loading ofRNA across all wells and between both gels. One Northern blot was probedwith a random primed P32 labelled probe encompassing the full codingregion of the α2 cDNA (from ATG to TAG) to assess α2 expression. Thesecond Northern blot was probed with a random primed P32 labelled probeencompassing the full coding region of the β10 cDNA (from ATG to TGA) toassess β10 expression.

Hybridization was for one hour at 65° C. in “ExpressHyb HybridizationSolution” (Clontech, Palo Alto, Calif.). The blots were washed in 2×SSC,0.1% SDS at room temperature twice, for twenty minutes each time. Theblots were then washed in 0.1×SSC, 0.1% SDS at 50° C. for ten minutes,and then exposed to film.

The results of the Northern analysis are as follows:

For the non-transgenic control mice no signal was found for either α2 orβ10.

For transgenic mouse #94 (male) no signal was found for either α2 orβ10.

For transgenic mice #s 25 (female) and 45 (female) a strong signal wasfound for both α2 and β10.

For transgenic mouse #95 (male) a moderate signal was found for both α2and β10.

For transgenic mouse #53 (male) a moderate signal was found for α2 and aweaker signal for β10.

For transgenic mouse #31 (male) a moderate signal was found for β10 butno signal was found for α2, indicating that mouse #31 overexpressed onlyβ10 and not α2. The level of β10 expression in mouse #31 wassignificantly greater than that found in mouse #53. The expressionresults for transgenic mouse #31 are consistent with the PCR genotypingdescribed above which for #31 was positive for the “mouse genomic β10human apoE expression cassette” but negative for the “mouse genomic α2human apoE expression cassette”. The data for mouse #31 indicates thatthe “mouse genomic α2 human apoE expression cassette” region of the“mouse genomic β10 mouse genomic α2 human apoE expression vectortransgene” DNA was truncated at some point during the microinjectionprocess resulting in a mouse which overexpresses β10 but not α2.Shearing/truncation of transgene DNA during the process of creatingtransgenic mice has been reported previously in the transgenicliterature.

H&E and BrdU stained sections of liver, gall bladder, spleen, lung,brain, pituitary, heart, kidney, adrenal, thymus, stomach, smallintestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammarygland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinarybladder, ovary or testis, uterus or prostate and seminal vesicle, bone,and bone marrow from the 4 α2/β10 overexpressors (#s 25, 45, 53 and 95),the 5 non-transgenic control mice (#s 17, 18, 19, 20 and 21), andtransgenic mouse #31, which only overexpressed β10 but not α2, wereexamined.

Immunohistochemical staining for BrdU was done on 4 μm thick paraffinembedded sections using an automated DPC Mark 5 Histochemical StainingSystem (Diagnostic Products Corp, Randolph, N.J.). Deparaffinized tissuesections were digested with 0.1% protease and then treated with 2Nhydrochloric acid. Sections were blocked with CAS BLOCK (ZymedLaboratories, San Francisco, Calif.), incubated with a rat anti-BrdUmonoclonal antibody (Accurate Chemical and Scientific, Westbury, N.Y.).The primary antibody was detected with a biotinylated rabbit anti-ratimmunoglobulin polyclonal antibody (Dako, Carpinteria, Calif.). Sectionswere then quenched with 3% hydrogen peroxide, and reacted with anavidin-biotin complex tertiary (Vector Laboratories). The stainingreaction was visualized with diaminobenzidine (DAB, Dako Carpinteria,Calif.) and sections were counterstained with hematoxylin.

All four α2/β10 overexpressors exhibited hepatomegaly (6.75±0.68% BW vs.4.98±0.29% BW in non-transgenic control mice, p=0.0011) and renalhypertrophy (2.23±0.21% BW vs. 1.75±0.12% BW in non-transgenic controlmice, p=0.0033). α2/β10 overexpressor mice also had a slightly lowermean body weight than their non-transgenic control counterparts; thisdifference was not statistically significant. α2/β10 overexpressor mice#s 45 and 53 also exhibited moderate splenomegaly. Transgenic mouse #31,which only overexpressed β10 and not α2, had normal liver, kidney andspleen weights.

All four α2/β10 overexpressor mice had elevated serum T4 levels(23.1±5.4 micrograms/dl vs. 5.0±0.7 micrograms/dl in non-transgeniccontrol mice, p=0.0001) and transgenic mice #s 25 and 45 had a modestcirculating lymphocytosis. Transgenic mouse #31, which onlyoverexpressed β10 and not α2, had normal serum T4 levels and lymphocytecounts. Individual serum T4 values for each mouse are as follows: #17(5.0 micrograms/dl), #18 (4.8 micrograms/dl), #19 (6.3 micrograms/dl),#20 (4.4 micrograms/dl), #21 (4.7 micrograms/dl), #25 (28.5micrograms/dl), #45 (26.9 micrograms/dl), #53 (18.2 micrograms/dl), #95(18.7 micrograms/dl), and #31 (3.2 micrograms/dl).

H&E and BrdU stained sections of liver, gall bladder, spleen, lung,brain, pituitary, heart, kidney, adrenal, thymus, stomach, smallintestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammarygland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinarybladder, ovary or testis, uterus or prostate and seminal vesicle, bone,and bone marrow from the four α2/β10 overexpressor mice, the 5non-transgenic control mice, and mouse #31, which only overexpressed β10and not α2, were examined. Histologically, all four α2/β10 overexpressormice exhibited bilaterally enlarged thyroid glands containing multiplefollicular papillary adenomas. All four α2/β10 overexpressor mice alsoexhibited mild to moderate hepatocellular hyperplasia with an increasein hepatocellular BrdU labeling vs. the non-transgenic mice. Transgenicmouse #31 had none of these features.

In summary, the four α2/β10 overexpressor transgenic mice exhibited aphenotype characterized by bilateral thyroid enlargement with multiplefollicular papillary adenomas and resulting hyperthyroidism, asindicated by elevated serum T4 levels. Other phenotypic changes werefelt to be related to the systemic hyperthyroid state, and includedmoderate hepatomegaly, hepatocellular hyperplasia, and slightlydecreased serum cholesterol levels, bilateral renal hypertrophy, and amild to moderate leukocytosis with a predominance of lymphocytes.

Transgenic mice over expressing mouse α2 and not β10 (#s 7, 8, 26, 28,156 and 186), described above, had no phenotype and transgenic mouse #31over expressing β10 and not α2 had no phenotype. This indicates that thehyperthyroid phenotype found in all four α2/β10 overexpressor transgenicmice (#s 25, 45, 53 and 95) can be attributed to the α2/β10heterodimeric hormone described in the present invention.

1. An isolated nucleic acid molecule encoding polypeptide comprising theamino acid sequence selected from the group consisting of: (a) the aminoacid sequence as set forth in SEQ ID NO:3, further comprising anamino-terminal methionine; (b) an amino acid sequence that is at least95% identical to the amino acid sequence of SEQ ID NO:3; (c) a fragmentof the amino acid sequence set forth in SEQ ID NO:3 comprising at leastabout 75 amino acid residues; (d) the amino acid sequence set forth inSEQ ID NO:3 having 1 to 25 conservative amino acid substitutions; (e)the amino acid sequence set forth in SEQ ID NO:3 having 1 to 25 aminoacids inserted; and (f) the amino acid sequence set forth in SEQ ID NO:3having 1 to 25 amino acids deleted wherein said polypeptide binds thealpha-2 subunit.
 2. A vector comprising the nucleic acid molecule ofclaim
 1. 3. A host cell comprising the vector of claim
 2. 4. The hostcell of claim 3 that is a eukaryotic.
 5. The host cell of claim 4 thatis a prokaryotic cell.
 6. A process of producing a polypeptidecomprising the amino acid sequence selected from the group consistingof: (a) the amino acid sequence as set forth in SEQ ID NO:3, furthercomprising an amino-terminal methionine; (b) an amino acid sequence thatis at least 95% identical to the amino acid sequence of SEQ ID NO:3; (c)a fragment of the amino acid sequence set forth in SEQ ID NO:3comprising at least about 75 amino acid residues; (d) the amino acidsequence set forth in SEQ ID NO:3 having 1 to 25 conservative amino acidsubstitutions; (e) the amino acid sequence set forth in SEQ ID NO:3having 1 to 25 amino acids inserted; and (f) the amino acid sequence setforth in SEQ ID NO:3 having 1 to 25 amino acids deleted, comprisingculturing the host cell of claim 3 under suitable conditions to expressthe polypeptide, and optionally isolating the polypeptide from theculture wherein said polypeptide binds the alpha-2 subunit.
 7. Theprocess of claim 6, wherein the nucleic acid molecule comprises humanApoE promoter DNA operatively linked to the nucleic acid encoding thepolypeptide.